Cardiac myosin light chain kinase-specific antibodies and methods of detecting

ABSTRACT

The present disclosure provides a cDNA, protein sequence, and genomic structure of the human cardiac isoform of myosin light chain kinase (cMLCK), and describes mutations in the cMLCK gene that are associated with cardiac dysfunction. Methods are provided for identifying individuals who can harbor mutations in the cMLCK gene, or carry alleles that can predisposed them to cardiac dysfunction. Disclosed also is a significant role for cMLCK in modulating cardiac contractility. The cMLCK protein is shown herein to reduce the amplitude of stretch activation and increase the tension production, a property of muscle which has heretofore had an unknown role in cardiac contraction. Moreover, the cMLCK protein is shown to be regionally distributed in the heart, thereby having differential effects on contractility and stretch activation. Methods herein are provided to exploit this effect of cMLCK, to treat individuals who have or are prone to cardiac dysfunction. In addition, methods are provided to identify agents that modulate cMLCK activity, thereby having potential therapeutic importance in the treatment of cardiac dysfunction.

PRIORITY CLAIM

This is a divisional of U.S. patent application Ser. No. 10/380,236,filed Sep. 25, 2003, which issued as U.S. Pat. No. 7,375,185. U.S.patent application Ser. No. 10/380,236 is a §371 U.S. national stage ofPCT/US01/28639, filed Sep. 12, 2001, which was published in Englishunder PCT Article 21(2), and claims the benefit of U.S. ProvisionalApplication No. 60/232,246, filed Sep. 12, 2000 and U.S. ProvisionalApplication No. 60/232,456, filed Sep. 13, 2000. U.S. patent applicationSer. No. 10/380,236 is incorporated by reference herein in its entirety.

BACKGROUND

The function of a mammal's circulatory system—its heart, lungs, bloodvessels and red blood cells—is to provide oxygen and nutrients to everycell. The heart's role is to pump oxygenated red blood cells to thetissues, to receive deoxygenated blood from the tissues, and to pumpdeoxygenated blood to the lungs where it can again take up oxygen. Heartfailure can be viewed as the failure to fulfill this role.

Heart failure affects more than 2 million Americans, and is a majorcause of illness, hospitalization, and death around the world. Currentlyavailable therapies includes medications such as digoxin and angiotensinconverting enzyme inhibitors, but these have had limited impact onmorbidity and mortality. Left ventricular assist devices show promise,but remain experimental. Cardiac transplantation is limited by ashortage of available hearts, and the need for permanentimmunosuppression. Thus, improved therapies for heart failure areneeded.

To develop improved therapies for heart failure, a more completeunderstanding of the heart's normal operation is needed. With this morecomplete understanding, specific aspects of heart function can betargeted for pharmacologic therapy, gene therapy, and other noveltherapeutic approaches.

Many basic facts about heart function are known. The heart is largelymade up of cardiac muscle, or myocardium. The myocardium mediates theheart's pumping function by automatically contracting and relaxing in acyclical manner. The contraction drives the blood forward, while therelaxation phase creates negative pressure that helps the heart to fillwith blood. This alternation of myocardial contraction and relaxation istermed the cardiac cycle.

In pumping blood, the vertebrate heart takes advantage of the increasedefficiency of wringing compared to compression. Just as both handsrotate in opposite directions during the squeeze, the base of the heartrotates in the clockwise direction as the apex rotatescounter-clockwise. An advantage of these mechanics is the reduction ofchamber volume and consequential decrease in wall stress. Theorientations of many cardiac muscle bundles facilitate the wringing aswell as compressing forces.

The molecular motor that drives contraction of cardiac muscle is myosin.The role of myosin is to transduce chemical energy into movement byhydrolyzing the high-energy phosphodiester bond of ATP.

Myosin is a large protein made up of three subunits, the myosin heavychain and two myosin light chains termed essential light chain (ELC) andregulatory light chain (RLC). The myosin heavy chain is an elongatedmolecule with a filamentous tail and a globular head. A “neck” regionlies between the tail and head. The tails self-assemble into filaments,with the myosin head extending outward from the filament. Thesemyosin-containing filaments are termed thick filaments. They interactwith thin filaments, which contain actin polymers. The actin polymersactivate the ATPase activity found in the myosin head. Movement isgenerated when the myosin heads: (1) bind to actin filaments; (2)hydrolyze ATP, thereby generating a lever like motion at the myosinneck; and (3) detach from sites on the actin-containing thin filament.The constant repetition of this cycle pushes the thin filament past thethick filaments, thereby generating differential motion. Multiplied overmillions of highly organized cardiac cells, the result is a highlycoordinated cycle of contraction and relaxation.

The trigger for cardiac contraction is a transient rise in theintracellular level of calcium. The actin-containing thin filament bindsan additional protein complex called troponin. In the absence ofcalcium, troponin interferes with the actin-myosin interaction. However,the troponin complex contains a high-affinity calcium binding proteinwhich binds calcium, thereby triggering a movement of the complex whichallows actin and myosin to interact productively. Cardiomyocytes containintracellular calcium stores that rapidly release calcium and take itback, thereby promoting the cycle of contraction and relaxation.

The neck region of the myosin heavy chain is supported by the two myosinlight chains. The precise role of these myosin light chains in cardiacmuscle has remained elusive. In smooth muscle (found in blood vesselsand internal organs, for example) the RLC plays a critical regulatoryrole: for contraction to proceed, the RLC must be phosphorylated by acalcium-activated enzyme called myosin light chain kinase (MLCK). In theabsence of MLCK-mediated RLC phosphorylation, smooth muscle myosinATPase activity is not activated, and the muscle remains relaxed.

In stark contrast to smooth muscle, cardiac RLC phosphorylation haslittle effect on myosin ATPase activity. A modest increase insensitivity to calcium has been described in isolated, chemically“skinned” (i.e., outer membranes removed) fibers in vitro, but thisobservation is of doubtful in vivo significance. Nevertheless, aphosphorylatable serine homologous to smooth muscle RLC has beenpreserved throughout evolution, and the reasons for this conservationhave remained a mystery.

Further study of a possible role for cardiac RLC phosphorylation hasbeen significantly hampered by the lack of sequence information aboutthe cardiac form of MLCK. What is needed is the complete cDNA sequenceof cardiac MLCK in humans and other mammalian species, as well as thededuced amino acid sequence and genomic sequence.

Indirect flight muscle (IFM) of insects has the same basic contractileapparatus as mammalian cardiac muscle: a myosin based thick filamentcomprised of myosin heavy and light chains; and an actin-containing thinfilament activated by calcium binding to troponin. However, IFM mustcontract and relax 150 times per second during flight. It would beenergetically wasteful to regulate this extraordinarily rapid cycleexclusively through release and reuptake of calcium from intracellularstores. Thus, IFM has evolved to accentuate and exploit a property ofmuscle contraction termed stretch activation.

The stretch activation response of IFM manifests itself as a “delayedtension” when an activated muscle fiber is subjected to a quick stretch.When tension is monitored as a function of time (for example, byattaching an isolated muscle to a sensitive force transducer), and IFMis quickly stretched, an immediate increase in tension is observed whichrapidly decays. This immediate tension increase is mediated by elasticrecoil. In IFM, there is a second, delayed rise in tension which isdefined as stretch activation. This response has been shown to be acritical component of IFM function, since it contributes substantiallyto oscillatory power output. Drosophila mutants lacking stretchactivation have no ability to fly.

The role of stretch activation can be likened to pushing a child on aswing: when a swing is at the rear of its arc, it has zero velocity andis about to be pulled forward by gravity. A properly timed push is avery efficient way to enhance the forward swinging force. In IFM,stretch activation corresponds to the push.

Stretch activation is intimately related to another important propertyof IFM, namely resonant frequency. As in the swing metaphor, the swingarc has a predictable frequency, and will return to the pushingindividual at a particular time. This predictable frequency is theswing's resonant frequency. The individual must time the push to theresonant frequency. Such precise timing will maximally enhance theswinging motion's amplitude with the least amount of effort. Animproperly timed push will not enhance the amplitude, and may in factwork against the swinging motion. Similarly, the resonant frequency ofstretch activation in IFM must be precisely matched to the cycle ofmuscle contraction and relaxation.

Several mutations in human cardiac ELCs and RLCs are associated with anunusual inherited disease of cardiac muscle (cardiomyopathy) termedmid-cavitary ventricular hypertrophy (MCVH; Poetter et al., NatureGenetics 13: 63-69, 1996). In its fully developed form, MCVH ischaracterized by massive overgrowth or hypertrophy largely confined tothe center of the left ventricle—the papillary muscles, and adjacentinterventricular septum and left ventricular free walls. The physiologicbasis for this unusual, regionally confined hypertrophy is unknown.Interestingly, however, when a mutant human cardiac ELC is expressed intransgenic mice, the mice develop regional hypertrophy indistinguishablefrom human MCVH. Papillary muscles removed from the hearts of thesetransgenic mice show altered stretch activation, even before thehypertrophy develops (Vermuri et al., PNAS 96: 1048-1053, 1999). Thealteration included a significantly increased resonant frequency.

It would be helpful to determine whether stretch activation has asignificant role in mammalian cardiac muscle, and to develop newtherapies for heart disease based on modulation of stretch activation.Improved and more comprehensive methods of identifying individuals atrisk of developing cardiac dysfunctions, such as cardiomyopathy, wouldalso be beneficial.

SUMMARY OF THE DISCLOSURE

The foregoing problems are addressed by the present invention, whereinthe cardiac myosin light chain kinase (cMLCK) gene of humans has beenidentified and cloned, the sequence of the cDNA and protein determined,and the role of cMLCK in cardiac contraction clarified. It is shownherein that cardiac myosin light chain kinase is regionally distributedin the heart, and is most active at the apex and base of the heart. Itis further shown that cMLCK phosphorylation of human cardiac RLCsurprisingly decreases the amplitude of the stretch activation response,thereby reducing the impact of stretch activation on cardiaccontraction.

The foregoing and other objects, features, and advantages of thecompositions and methods disclosed herein will become more apparent fromthe following detailed description of several embodiments, whichproceeds with reference to the accompanying sequence listing.

Sequence Listing

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. In theaccompanying sequence listing:

SEQ ID NO: 1 shows the nucleotide sequence of the cMLCK cDNA;

SEQ ID NO: 2 shows the amino acid sequence of the cMLCK protein;

SEQ ID NOs: 3-14 show exons 1-12 of the cMCLK, with surrounding intronsequences.

SEQ ID NO: 15 shows the sequence of the peptide used to generateantibody to the phosphorylated form of human RLC.

SEQ ID NO: 16 shows the amino acid sequence of a human cardiac MLCpeptide.

SEQ ID NOs: 17-24 are primer sequences.

SEQ ID NO: 25 is the c-terminal 46 residues of cMLCK.

SEQ ID NO: 26 is a peptide for producing antibodies.

DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments disclosedherein, the following list of abbreviations and definition of terms isprovided:

I. Abbreviations and Definitions

A. Abbreviations

ATP: Adenosine triphosphate

ELC: essential myosin light chain; also referred to as MLC1

HCM: hypertrophic cardiomyopathy

MVC: mid-ventricular cavitary hypertrophy

cMLCK: cardiac isoform of myosin light chain kinase

MLCK: myosin light chain kinase

MLC: myosin light chain

RLC: regulatory myosin light chain, also referred to as MLC2

RLC-P: phosphorylated form of RLC, after phosphorylation by MLCK.

IFM: indirect flight muscle of insects

P1: P1-derived artificial chromosome

PCR: polymerase chain reaction

B. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VI, published by Oxford UniversityPress, 1997 (ISBN 0-19-857778-8); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Amplification: When used in reference to a nucleic acid, techniques thatincreases the number of copies of a nucleic acid molecule in a sample orspecimen. An example of amplification is the polymerase chain reaction,in which a biological sample collected from a subject is contacted witha pair of oligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification can be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse compliment), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget.

Binding or stable binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including both functional and physicalbinding assays. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription,translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it isso simple and reliable, involves observing a change in light absorptionof a solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target disassociate from each other, ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

Cardiac: pertaining to the heart.

Cardiac myosin light chain kinase gene: A novel gene that codes for acardiac MLCK protein, the mutation of which is associated withhereditary increased susceptibility to cardiac dysfunction. A mutationof the cardiac MLCK gene can include nucleotide sequence changes,additions or deletions, including deletion of large portions or theentire cardiac MLCK gene, or duplications of all or substantially all ofthe gene. Alternatively, genetic expression of cardiac MLCK can bederegulated such that cardiac MLCK is over or under expressed. The term“cardiac MLCK gene” is understood to include the various sequencepolymorphisms and allelic variations that exist within the population.This term relates primarily to an isolated coding sequence, but can alsoinclude some or all of the flanking regulatory elements and/or intronsequences.

Mutant forms and altered expression of the cardiac MLCK gene can beassociated with hereditary cardiomyopathy. The RNA transcribed from amutant cardiac MLCK gene is mutant cardiac MLCK messenger RNA.

Cardiac MLCK cDNA: A cDNA molecule which, when transfected or otherwiseintroduced into cells, expresses the cardiac MLCK protein. The cardiacMLCK cDNA can be derived, for instance, by reverse transcription fromthe mRNA encoded by the cardiac MLCK gene and lacks internal non-codingsegments and transcription regulatory sequences present in the cardiacMLCK gene. The prototypical human cardiac MLCK cDNA is shown in SEQ IDNO: 1.

Cardiac dysfunction: any impairment in the heart's pumping function.This includes, for example, impairments in contractility, impairments inability to relax (sometimes referred to as diastolic dysfunction),abnormal or improper functioning of the heart's valves, diseases of theheart muscle (sometimes referred to as cardiomyopathy), diseases such asangina and myocardial infarction characterized by inadequate bloodsupply to the heart muscle, infiltrative diseases such as amyloidosisand hemochromatosis, global or regional hypertrophy (such as may occurin some kinds of cardiomyopathy or systemic hypertension), and abnormalcommunications between chambers of the heart (for example, atrial septaldefect). For further discussion, see Braunwald, Heart Disease: aTextbook of Cardiovascular Medicine, 5th edition 1997, WB SaundersCompany, Philadelphia Pa. (hereinafter Braunwald).

Cardiomyopathy: any disease or dysfunction of the myocardium (heartmuscle). These can be inflammatory, metabolic, toxic, infiltrative,fibroplastic, hematological, genetic, or unknown in origin. They aregenerally classified into three groups based primarily on clinical andpathological characteristics:

-   -   (1) dilated cardiomyopathy, a syndrome characterized by cardiac        enlargement and impaired systolic function of one or both        ventricles;    -   (2) hypertrophic cardiomyopathy, herein defined as (a) global or        regional increase in thickness of either ventricular wall or the        interventricular septum, or (b) an increased susceptibility to        global or regional increase in thickness of either ventricular        wall or the interventricular septum, such as can occur in        genetic diseases, hypertension, or heart valve dysfunction; or    -   (3) restrictive and infiltrative cardiomyopathies, a group of        diseases in which the predominate clinical feature is usually        impaired ability of the heart to relax (diastolic dysfunction),        and often characterized by infiltration of the heart muscle with        foreign substances such as amyloid fibers, iron, or glycolipids.

See Wynne and Braunwald, The Cardiomyopathies and Myocarditities,Chapter 41 in Braunwald.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA canalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA issynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

DNA: deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide. Theterm codon is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Deletion: The removal of a sequence of DNA, the regions on either sidebeing joined together.

Effective amount of a compound: A quantity of compound sufficient toachieve a desired effect in a subject being treated. For instance, thiscan be the amount necessary to increase the heart rate or cardiaccontractility of the subject. In general, this amount will be sufficientto measurably increase the number of beats per minute of the heart, orsufficient to increase cardiac contractility in some measurable way,such as by echocardiography, measurement of cardiac output, orimprovement in signs or symptoms of congestive heart failure.

An effective amount of a compound can be administered in a single dose,or in several doses, for example daily, during a course of treatment.However, the effective amount of the compound will be dependent on thecompound applied, the subject being treated, the severity and type ofthe affliction, and the manner of administration of the compound.

The general term “administering to the subject” is understood to includeall animals (e.g. humans, apes, dogs, cats, horses, and cows) that haveor may develop some form of cardiac dysfunction.

Electrical pacing: controlling or attempting to control the rate atwhich the heart beats by external electrical stimulation. See Barold etal., Cardiac Pacemakers and Antiarrythmic Devices, Chapter 23 inBraunwald for a more detailed discussion.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Functional fragments and variants of a polypeptide: includes thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more the polypeptide's functions. First, the genetic code iswell-known to be degenerate, and thus different codons encode the sameamino acids. Second, even where an amino acid substitution isintroduced, the mutation can be conservative and have no material impacton the essential functions of a protein. See Stryer, Biochemistry 3rdEd., (c) 1988. Third, part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. Fourth, insertions oradditions can be made in the polypeptide chain—for example, addingepitope tags—without impairing or eliminating its functions (Ausubel etal., 1997). Other modifications that can be made without materiallyimpairing one or more functions of a polypeptide include, for example,in vivo or in vitro chemical and biochemical modifications or whichincorporate unusual amino acids. Such modifications include, forexample, acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, labeling, e.g., with radionuclides, and various enzymaticmodifications, as will be readily appreciated by those well skilled inthe art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ³²P, ligands which bind tolabeled antiligands (e.g., antibodies), fluorophores, chemiluminescentagents, enzymes, and antiligands. Functional fragments and variants canbe of varying length. For example, some fragments have at least 10, 25,50, 75, 100, or 200 amino acid residues.

A functional fragment or variant of myosin light chain is defined hereinas a polypeptide which is capable of being phosphorylated by a proteinhaving myosin light chain kinase biological activity. It includes anypolypeptide six or more amino acid residues in length which is capableof being phosphorylated by a protein having myosin light chain kinasebiological activity.

Heart: the muscular organ of an animal that circulates blood.

In mammals, the heart is comprised of four chambers: right atrium, rightventricle, left atrium, left ventricle. The right atrium and left atriumare separated from each other by an interatrial septum, and the rightventricle and left ventricle are separated from each other by aninterventricular septum. The right atrium and right ventricle areseparated from each other by the tricuspid valve. The left atrium andleft ventricle are separated from each other by the mitral valve.

The walls of the heart's four chambers are comprised of working muscle,or myocardium, and connective tissue. Myocardium is comprised ofmyocardial cells, which are also referred to herein as cardiac cells,cardiac myocytes, cardiomyocytes and/or cardiac fibers. Myocardial cellscan be isolated from a subject and grown in vitro. The inner layer ofmyocardium closest to the cavity is termed endocardium, and the outerlayer of myocardium is termed epicardium. The left ventricular cavity isbounded in part by the interventricular septum and the left ventricularfree wall. The left ventricular free wall is sometimes divided intoregions, such as anterior wall, posterior wall and lateral wall; or apex(the tip of the left ventricle, furthest from the atria) and base (partof the left ventricle closest to the atria). Apical and basal areadjectives that refer to the corresponding region of the heart.

In operation, the heart's primary role is to pump sufficient oxygenatedblood to meet the metabolic needs of the tissues and cells in a subject.The heart accomplishes this task in a rhythmic and highly coordinatedcycle of contraction and relaxation referred to as the cardiac cycle.For simplicity, the cardiac cycle may be divided into two broadcategories: ventricular systole, the phase of the cardiac cycle wherethe ventricles contract; and ventricular diastole, the phase of thecardiac cycle where the ventricles relax. See Opie, Chapter 12 inBraunwald for a detailed discussion. Used herein, the terms systole anddiastole are intended to refer to ventricular systole and diastole,unless the context clearly dictates otherwise.

In normal circulation during health, the right atrium receivessubstantially deoxygenated blood from the body via the veins. Indiastole, the right atrium contracts and blood flows into the rightventricle through the tricuspid valve. The right ventricle fills withblood, and then contracts (systole). The force of systole closes thetricuspid valve and forces blood through the pulmonic valve into thepulmonary artery. The blood then goes to the lungs, where it releasescarbon dioxide and takes up oxygen. The oxygenated blood returns to theheart via pulmonary veins, and enters the left atrium. In diastole, theleft atrium contracts and blood flows into the left ventricle throughthe mitral valve. The left ventricle fills with blood and thencontracts, substantially simultaneously with right ventricularcontraction. The force of contraction closes the mitral valve and forcesblood through the aortic valve into the aorta. From the aorta,oxygenated blood circulates to all tissues of the body where it deliversoxygen to the cells. Deoxygenated blood then returns via the veins tothe right atrium.

In the cavity of left ventricle, there are two large, essentiallycone-shaped extensions of the ventricular myocardium known as theanterior and posterior papillary muscles. These connect to theventricular surface of the mitral valve via threadlike extensions termedchordae tendiniae or chordae. One important role for the papillarymuscles and chordae is to ensure that the mitral valve stays closedduring ventricular systole. Another important role is to add to theforce of cardiac contraction. Similarly, the right ventricle haspapillary muscles and chordae which tether the tricuspid valve and addto the force of contraction.

Due to inherited or acquired disease processes and/or normal aging, theheart muscle can develop dysfunction of either systole or diastole, orboth. Dysfunction of systole is referred to as systolic dysfunction.Dysfunction of diastole is referred to as diastolic dysfunction. SeeOpie Chapter 12, and Colucci et al., Chapter 13 in Braunwald for adetailed discussion.

Due to inherited or acquired disease processes and/or normal aging, oneor more of the heart valves may develop dysfunction. Valvulardysfunction generally falls into two broad categories: stenosis, definedherein as incomplete opening of the valve during a time of the cardiaccycle when a normally operating valve is substantially open; andinsufficiency, defined herein as incomplete closing of the valve duringa time of the cardiac cycle when a normally operating valve issubstantially closed. Valvular dysfunction also includes a conditionknown as mitral valve prolapse, wherein the mitral valve leafletsprolapse backward into the left atrium during ventricular systole. Thecondition may be associated with mild, moderate, or severe insufficiencyof the mitral valve.

Valvular stenosis is typically characterized by a pressure gradientacross the valve when the valve is open. Valvular insufficiency istypically characterized by retrograde (“backward”) flow when the valveis closed. For example, mitral stenosis is characterized by a pressuregradient across the mitral valve near the end of ventricular diastole(as a typical example of moderate mitral stenosis, 5 mm Hg diastolicpressure in the left ventricle, 20 mm Hg diastolic pressure in the leftatrium, for a pressure gradient of 15 mm Hg). As another example, mitralinsufficiency is characterized by “backward” flow of blood from the leftventricle into the left atrium during ventricular systole.

Heart failure: the inability of the heart to supply sufficientoxygenated blood to meet the metabolic needs of the tissues and cells ina subject. This can be accompanied by circulatory congestion, such ascongestion in the pulmonary or systemic veins. As used herein, the termheart failure encompasses heart failure from any cause, and is intendedherein to encompass terms such as “congestive heart failure,” “forwardheart failure,” “backward heart failure,” “high output heart failure,”“low output heart failure,” and the like. See Chapters 13-17 inBraunwald for a detailed discussion.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Labeled: a biomolecule attached covalently or noncovalently to adetectable label or reporter molecule. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, e.g., in Sambrook et al., MolecularCloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publ. Assoc. andWiley-Intersciences, 1998. For example, ATP can be labeled in any one ofits three phosphate groups with radioisotopes such as ³²P or ³³P, or inits sugar moiety with a radioisotopes such as ³⁵S.

Myosin light chain: an approximately 18 kDa protein which associateswith the myosin heavy chain and participates in the regulation ofmyosin's force-generating ATPase activity. There are two major groupingsof MLC: MLC1, sometimes referred to as the essential myosin light chain,abbreviated ELC; and MLC2, sometimes referred to as the regulatorymyosin light chain, abbreviated RLC. RLC is the primary biologicaltarget of MLCK-mediated phosphorylation. When phosphorylated by MLCK thephosphorylated form of RLC is abbreviated RLC-P. Isoforms of ELC and RLCexisting in skeletal, smooth, and cardiac muscle have been described. Asan example, the human cardiac RLC gene and cDNA are described by Maceraet al., Genomics 13: 829-31, 1992 (GenBank accession no. NM00432).

Myosin light chain kinase biological activity: the in vitro or in vivoenzymatic ability of a polypeptide or protein to mediate covalentincorporation of a phosphate into a regulatory myosin light chain. Theterm encompasses such enzymatic activity observed with any isoform ofMLCK (for example, nonmuscle, smooth muscle, skeletal muscle, andcardiac MLCK isoforms), as well as such enzymatic activity observed withfragments and variants of MLCK isoforms (for example, naturallyoccurring mutants; mutations, insertions and deletions introducedthrough recombinant DNA techniques; and fragments of MLCK generated byproteolysis).

Muscle cell: include skeletal, cardiac or smooth muscle tissue cells.This term is synonymous with myocyte, and encompasses those cells whichdifferentiate to form more specialized muscle cells (e.g. myoblasts).“Cardiomyocyte” refers to a cardiac muscle cell, or cells thatdifferentiate to form cardiomyocytes.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Ortholog: Two nucleic acid or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species.Orthologous sequences are also homologous sequences.

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on the nucleic acid molecules provided in this invention.A probe comprises an isolated nucleic acid attached to a detectablelabel or reporter molecule. Typical labels include radioactive isotopes,enzyme substrates, co-factors, ligands, chemiluminescent or fluorescentagents, haptens, and enzymes. Methods for labeling and guidance in thechoice of labels appropriate for various purposes are discussed, e.g.,in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989) and Ausubel et al. (In Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).

Primers are short nucleic acid molecules, preferably DNAoligonucleotides 10 nucleotides or more in length. More preferably,longer DNA oligonucleotides can be about 15, 17, 20, or 23 nucleotidesor more in length. Primers can be annealed to a complementary target DNAstrand by nucleic acid hybridization to form a hybrid between the primerand the target DNA strand, and then the primer extended along the targetDNA strand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (In Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1998), and Inniset al. (PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990). PCR primer pairs can be derivedfrom a known sequence, for example, by using computer programs intendedfor that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 30 consecutive nucleotides of the cardiac MLCK encodingnucleotide will anneal to a target sequence, such as another cardiacMLCK gene homolog from the gene family contained within a human genomicDNA library, with a higher specificity than a corresponding primer ofonly 15 nucleotides. Thus, in order to obtain greater specificity,probes and primers can be selected that comprise at least 17, 20, 23,25, 30, 35, 40, 45, 50 or more consecutive nucleotides of cardiac MLCKnucleotide sequences.

The invention thus includes isolated nucleic acid molecules thatcomprise specified lengths of the disclosed cardiac MLCK cDNA sequences.Such molecules can comprise at least 17, 20, 23, 25, 30, 35, 40, 45 or50 consecutive nucleotides of these sequences, and can be obtained fromany region of the disclosed sequences. By way of example, the cardiacMLCK cDNA sequences can be apportioned into halves or quarters based onsequence length, and the isolated nucleic acid molecules can be derivedfrom the first or second halves of the molecules, or any of the fourquarters. By way of example, the human cardiac MLCK cDNA, ORF, codingsequence and gene sequences can be apportioned into about halves orquarters based on sequence length, and the isolated nucleic acidmolecules (e.g., oligonucleotides) can be derived from the first orsecond halves of the molecules, or any of the four quarters. The humancardiac MLCK cDNA (SEQ ID NO: 1) can be used to illustrate this. Thehuman cardiac MLCK cDNA is 18207 nucleotides in length and so can behypothetically divided into about halves (nucleotides 1-9103 and9104-18207) or about quarters (nucleotides 1-4551, 4552-9103, 9104-13464and 13465-18207). The cDNA also could be divided into smaller regions,e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, withsimilar effect.

Alternatively, the coding sequence of the human cardiac MLCK cDNA can bethus apportioned into about halves or quarters, and oligonucleotidesderived from any such portion. The coding sequence of cardiac MLCK is16190 nucleotides in length, and corresponds to nucleotides 230-17140 ofthe cDNA (SEQ ID NO: 1). The coding sequence thus can be hypotheticallydivided into about halves (nucleotides 1-8455 and 8456-16910 of thecoding sequence, corresponding to positions 230-8685 and 8686-17140,respectively, of SEQ ID NO: 1) or about quarters (nucleotides 1-4227,4228-8455, 8465-12683 and 12683-16190 of the coding sequence,corresponding to positions 230-4457, 4458-8685, 8686-12913, and12914-17140, respectively, of SEQ ID NO: 1). The coding sequence ofcardiac MLCK also could be divided into smaller regions, e.g. abouteighths, sixteenths, twentieths, fiftieths and so forth, with similareffect.

Protein: A biological molecule expressed by a gene and comprised ofamino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of the cardiac MLCKprotein, and the corresponding cDNA sequence, will possess a relativelyhigh degree of sequence identity when aligned using standard methods.This homology will be more significant when the orthologous proteins orcDNAs are derived from species which are more closely related (e.g.,human and chimpanzee sequences), compared to species more distantlyrelated (e.g., human and C. elegans sequences).

Typically, cardiac MLCK orthologs are at least 50% identical at thenucleotide level and at least 50% identical at the amino acid level whencomparing human cardiac MLCK to an orthologous cardiac MLCK.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman (1981) Adv. Appl. Math. 2: 482; Needleman & Wunsch (1970) J.Mol. Biol. 48: 443; Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA85: 2444; Higgins & Sharp (1988) Gene, 73: 237-244; Higgins & Sharp(1989) CABIOS 5: 151-153; Corpet et al. (1988) Nuc. Acids Res. 16,10881-90; Huang et al. (1992) Computer Appls. in the Biosciences 8,155-65; and Pearson et al. (1994) Meth. Mol. Bio. 24, 307-31. Altschulet al. (1990) J. Mol. Biol. 215:403-410, presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.(1990) J. Mol. Biol. 215:403-410) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program.

Homologs of the disclosed human cardiac MLCK protein typically possessat least 60% sequence identity counted over full-length alignment withthe amino acid sequence of human cardiac MLCK using the NCBI Blast 2.0,gapped blastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 75% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are described in the NCBIwebsite.

These sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided. The present invention providesnot only the peptide homologs that are described above, but also nucleicacid molecules that encode such homologs.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL,New York and Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York. Nucleic acid molecules that hybridize understringent conditions to a human cardiac MLCK gene sequence willtypically hybridize to a probe based on either an entire human cardiacMLCK gene or selected portions of the gene under wash conditions of2×SSC at 50° C. A more detailed discussion of hybridization conditionsis presented below.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a cardiac MLCK protein-specific binding agent bindssubstantially only the cardiac MLCK protein. As used herein, the term“cardiac MLCK protein specific binding agent” includes anti-cardiac MLCKprotein antibodies and other agents (such as soluble receptors) thatbind substantially only to the cardiac MLCK protein.

Anti-cardiac MLCK protein antibodies can be produced using standardprocedures described in a number of texts, including Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Thedetermination that a particular agent binds substantially only to thecardiac MLCK protein can readily be made by using or adapting routineprocedures. One suitable in vitro assay makes use of the Westernblotting procedure (described in many standard texts, including Harlowand Lane, Antibodies, A Laboratory Manual, CSHL, New York, 1988).Western blotting can be used to determine that a given cardiac MLCKprotein binding agent, such as an anti-cardiac MLCK protein monoclonalantibody, binds substantially only to the cardiac MLCK protein.

A phosphospecific binding agent specifically binds to a peptidecontaining a phosphorylated residue.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto cardiac MLCK would be cardiac MLCK-specific binding agents. Theseantibody fragments are defined as follows: (1) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody moleculeproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (3) (Fab′)₂, the fragment of the antibody obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)2, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The object of identifying the hitherto unknown cMLCK gene has beenachieved by providing an isolated human cDNA molecule and genomic DNAstructure. Specifically, the disclosure provides, for the first time, anisolated cDNA molecule which, when transfected into cells can producethe cMLCK protein linked to cardiac dysfunction, cardiac hypertrophy andcertain forms of cardiomyopathy such as hypertrophic cardiomyopathy andmid-cavitary ventricular hypertrophy. The disclosure encompasses thecMLCK cDNA molecule, the nucleotide sequence of this cDNA, and theputative amino acid sequence of the cMLCK protein encoded by this cDNA.

Having herein provided the nucleotide sequence of the cMLCK cDNA,correspondingly provided are the complementary DNA strands of the cDNAmolecule and DNA molecules which hybridize under stringent conditions tothe cMLCK cDNA molecule or its complementary strand. Such hybridizingmolecules include DNA molecules differing only by minor sequencechanges, including nucleotide substitutions, deletions and additions.Also comprehended by this invention are isolated oligonucleotidescomprising at least a segment of the cDNA molecule or its complementarystrand, such as oligonucleotides, which can be employed as effective DNAhybridization probes or primers useful in the polymerase chain reaction.Such probes and primers are particularly useful in the screening anddiagnosis of persons genetically predisposed to hypertrophiccardiomyopathy and other forms of cardiac dysfunction, as the result ofcMLCK gene mutations. Generally, these oligonucleotides will be 10contiguous nucleotides long or longer, and preferably 20 contiguousnucleotides long or longer, and can be at least 25, 30, 35, 40, 45, or50 contiguous nucleotides in length.

Recombinant DNA vectors comprising the disclosed DNA molecules, andtransgenic host cells containing such recombinant vectors, are alsoprovided. Disclosed embodiments include transgenic nonhuman animalswhich over- or under-express the cMLCK protein, or over- orunder-express fragments or variants of cMLCK protein.

A disclosed embodiment is a method for screening a subject to determineif the subject carries a mutant cMLCK gene, or if the gene has beenpartially or completely deleted or duplicated. The method includes thesteps of: providing a biological sample obtained from the subject, whichsample includes DNA or RNA, and providing an assay for detecting in thebiological sample the presence of a mutant cMLCK gene, a mutant cMLCKRNA, or the absence, through partial or complete deletion, of the cMLCKgene and corresponding RNA, or the presence of multiple copies of thecMLCK encoding region through duplication.

The foregoing assay can be assembled in the form of a diagnostic kit andin some embodiments includes: hybridization with oligonucleotides; PCRamplification of the cMLCK gene or a part thereof using oligonucleotideprimers; RT-PCR amplification of the cMLCK RNA or a part thereof usingoligonucleotide primers; or direct sequencing of the cMLCK gene of thesubject's genome using oligonucleotide primers. The efficiency of thesemolecular genetic methods permits a rapid classification of patientsaffected by deletions or mutations of the cMLCK gene.

A further aspect of the present disclosure is a method for screening asubject to assay for the presence of a mutant, or partially or entirelyduplicated or deleted cMLCK gene, by providing a biological sample ofthe subject which sample contains cellular proteins, and providing animmunoassay for quantitating the level of cMLCK protein in thebiological sample, or a level of activity of the protein. Diagnosticmethods for the detection of mutant, duplicated or deleted cMLCK genesmade possible by this invention, or the detection of abnormal proteinfunction or expression, provides an enhanced ability to diagnosesusceptibility to hereditary cardiac dysfunction.

Another aspect of the disclosure is a preparation comprising one or morebinding agents that specifically detect the cMLCK protein. Such specificbinding agents can be antibodies, for instance monoclonal antibodies orpolyclonal antibodies. In addition, the invention provides specificbinding agents for the human regulatory myosin light chain in itsMLCK-phosphorylated form.

Also disclosed is a method for detecting enhanced susceptibility of asubject to cardiac dysfunction, by detecting decreased, increased, ormutant cMLCK in the subject's cells, such as cardiac or muscle cells.Enhanced susceptibility to dysfunction can also be detected bytransforming a cell with cDNA encoding cMLCK from the subject,expressing the cMLCK, and evaluating its myosin light chain kinasebiological activity.

The disclosure also provides methods for enhancing or preserving thecardiac function of a subject, by modulating the subject's cardiacstretch activation, for example, by modulating myosin phosphorylation orthe cMLCK biological activity in the myocardial cells of the subject.Disclosed methods for modulating cMLCK biological activity include, forexample, administering to the subject an effective amount of a compoundthat modulates cMLCK activity, or delivering to the subject's heart avector encoding a peptide that modulates cMLCK activity. The vector canbe delivered, for example, to specific regions of the heart, such aspapillary muscle or left ventricular free wall. The methods can be usedto treat cardiac dysfunction, for example, systolic dysfunction,diastolic dysfunction, cardiac hypertrophy, cardiomyopathy, coronaryheart disease, myocardial infarction, and congestive heart failure.

Methods are further provided for screening for agents which can modulatecMLCK biological activity, by incubating a putative modulator agent witha protein having cMLCK biological activity and a polypeptide which canserve as a substrate for that protein, and detecting phosphorylation ofthe polypeptide. Two or more concentrations of the putative modulatoryagent can be compared for their ability to modulate cMLCK activity, bycomparing an extent of polypeptide phosphorylation at the twoconcentrations. Alternatively, two or more different putative modulatoryagents can also be compared. Phosphorylation of the peptide can bedetected with a specific binding agent which specifically binds thephosphorylated form of the polypeptide, or by detecting incorporation oflabeled phosphate into the polypeptide. A secondary specific bindingagent, which can be labeled, can be used to detect the specificallybound primary specific binding agent.

II. cMLCK Protein and Nucleic Acid Sequences

This invention provides cMLCK proteins and cMLCK nucleic acid molecules,including cDNA sequences. The prototypical cMLCK sequences are the humansequences, and the invention provides for the use of these sequences toproduce transgenic animals having increased or decreased levels of cMLCKprotein, as well as diagnostic methods to detect defects or alterationsin cMLCK expression or cMLCK protein production. Also provided is thehuman cMLCK genomic structure and sequence.

The full-length cDNA for cMLCK is 1791 base pairs long, and encodes aprotein of 596 amino acids (SEQ ID NO: 1 and SEQ ID NO:2).

Cloning and Sequence Determination of the cMLCK cDNA and Genomic DNAfrom Rabbit Heart

Since the first report that cardiac myosin RLC is phosphorylated invivo, there has been an unsuccessful search for the responsible kinase.Because slow skeletal muscle myosin and its RLC are the primary cardiacventricular isoforms in many animals, the skeletal MLCK was consideredto be the likely responsible kinase in heart. However, attempts todemonstrate skeletal MLCK in the heart have been unsuccessful.

Published rabbit skeletal muscle MLCK cDNA sequence was used to design aset of primer pairs to amplify unique fragments from both rabbitskeletal muscle and cardiac RNA. A product from one pair of primers(upstream 5′-TGATCCAGCTGTACGCAGCC-3″ (SEQ ID NO: 19), downstream5′-CTTGAGGTCCAGGTGCAGC-3′ (SEQ. ID NO:20)) yielded identically sized 201bp fragments from both templates. Subcloning and sequencing showedidentical sequences suggesting that either skeletal muscle MLCK or apartially homologous isoform could be found in rabbit cardiac muscle. Apossible genomic contamination was excluded since the same primerscrossed an intron-exon boundary and generated a greater than 500 bp sizefragment from rabbit genomic DNA. The divergence of the latter genomicsequence from the cDNA sequence marked what was later found to be thehomologue of the human intron-exon-6 boundary.

Cloning and Sequence Determination of the Human cMLCK cDNA and GenomicDNA

Next, human genomic DNA was used as a template from which a MLCKfragment was amplified using primers derived from the rabbit sequence.In order to avoid cross-reaction with human smooth muscle MLCK sequence,the DNA sequence flanking the homologous intron-6 insertion point ofrabbit skeletal and smooth muscle MLCK was compared. A downstream regionof amino acid divergence was identified in the presumed region ofskeletal MLCK exon 7. The nucleotide sequence encoding this stretch wasthen compared for differences between rabbit and rat skeletal muscleMLCK cDNA sequence. A degenerate primer was prepared that encoded bothrat and rabbit sequence as well as some possible 3^(rd) position codonchanges. (5′-AGGTCCAg/aGTGCAGc/a/t/gACCCg/tCA-3′ (SEQ ID NO: 21))Upstream primers in presumed human exon 6 that were divergent betweenrabbit smooth and skeletal muscle MLCK were conserved between rat andrabbit skeletal MLCK sequence. Thus, minimal changes from the rabbitupstream primer sequence were made (5′-CGTg/cCTGTTCATGGAGT-3′ (SEQ IDNO: 22)). Using the latter 2 primers, the fragment obtained from humangenomic DNA contained an 82 bp intron. Subcloning and sequencing yieldedcoding sequence, which internal to the primer ends, showed significanthomology at the amino acid level to rabbit skeletal muscle MLCK.

In order to obtain a full length clone from human cardiac RNA, 5′ and 3′RACE was performed using the Marathon RACE kit (Clontech). The exon 6sequence obtained from human DNA was used to generate 2 primers for 5′and 3′ RACE. The 5′ RACE fragment was denatured and annealed to thehuman exon 6 containing fragment. PCR amplification using primers fromthe 5′ ends of both fragments were used to join both fragments. Asimilar process was then used to join this fragment with the 3′ RACEproduct to produce a full length cDNA fragment. This full length cDNAwas sequenced and matched the sequence of the RT-PCR amplified productfrom human skeletal muscle.

In order to obtain full length genomic sequence, a primer based in humanintron 6 (5′-CCACGGCTTGCTCCGTGCCT-3′ (SEQ ID NO: 23)) was used togetherwith an upstream exon 6 primer (5′-ATCGAGACTCCGCATGAGAT-3′ (SEQ ID NO:24)) to screen a human P1 library (Genome Systems). Intron-exonboundaries were established by amplifying the intervening introns usingcDNA sequence derived primers as well as direct sequencing of the P1clone. Sequence of the coding portions of the genomic clone matched thefull length cDNA sequence obtained through RACE. There was significanthomology between the predicted amino acid translation of the human cMLCKsequence and rabbit skeletal muscle MLCK sequence. However, amino acidsequence divergence was substantial in the amino-terminal end.

The genomic DNA of human cMLCK comprises 12 exons.

The following examples help illustrate the specific applications of thistechnology.

EXAMPLE 1 Method of Making cMLCK Encoding Sequences

The foregoing discussion describes the original means by which the cMLCKcDNAs were obtained and also provides the nucleotide sequence of theseclones. It also describes the genomic structure and sequence of rabbitand human cMLCK. With the provision of this sequence information, thepolymerase chain reaction (PCR) or other similar amplificationtechniques can be used in a more direct and simple method for producingcMLCK encoding sequences.

Total RNA is extracted from human cells by any one of a variety ofmethods well known to those of ordinary skill in the art. Sambrook etal. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989)and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1998) provide descriptions of methods for RNAisolation. In one embodiment, human myocardial cells obtained from amyocardial biopsy, or cultured human myocytes from a non-cMLCK deletedindividual are utilized. The extracted RNA is then used as a templatefor performing the reverse transcription-polymerase chain reaction(RT-PCR) amplification of cDNA. Methods and conditions for RT-PCR aredescribed in Kawasaki et al., In PCR Protocols, A Guide to Methods andApplications, Innis et al. (eds.), 21-27, Academic Press, Inc., SanDiego, Calif., 1990. The selection of PCR primers is made according tothe portions of the cDNA which are to be amplified. Primers are chosento amplify small segments of a cDNA or the entire cDNA molecule.Variations in amplification conditions are required to accommodateprimers and amplicons of differing lengths and composition; suchconsiderations are well known in the art and are discussed in Innis etal. (PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). cMLCK encoding sequences can beamplified theoretically using the following combination of primers:

primer 1 5′ atg gcg aca gaa aat gg 3′ (SEQ ID NO: 17) primer 3 5′ tcagac ccc cag agc ca 3′. (SEQ ID NO: 18)These primers are illustrative only; one skilled in the art willappreciate that many different primers can be derived from the providedcDNA sequence in order to amplify particular regions of these cDNAs.

Re-sequencing of PCR products obtained by these amplification proceduresis performed; this facilitates confirmation of the amplified sequenceand also provides information on natural variation on this sequence indifferent populations or species. Oligonucleotides derived from theprovided cMLCK sequences provided are used in such sequencing methods.

Orthologs of human cMLCK are cloned in a similar manner, where thestarting material consists of myocytes or cardiomyocytes taken from anon-human species. Orthologs will generally share at least 50% sequencehomology with the disclosed human cMLCK cDNA. Where the non-humanspecies is more closely related to humans, the sequence homology isgenerally greater. Closely related orthologous cMLCK molecules can shareat least 75%, at least 80%, at least 90%, at least 95%, or at least 98%sequence homology with the disclosed human sequences.

Oligonucleotides derived from the human cMLCK cDNA sequence (SEQ ID NO:1), are encompassed within the scope of the present invention. Sucholigonucleotide primers can for example include a sequence of at least10 consecutive nucleotides of the cMLCK nucleic acid sequence. Toenhance amplification specificity, oligonucleotide primers comprising atleast 15, 25, 30, 35, 40, 45 or 50 or more consecutive nucleotides ofthese sequences can also be used. These primers for instance can beobtained from any region of the disclosed sequences. By way of example,the human cMLCK cDNA, ORF and gene sequences may be apportioned intoabout halves or quarters based on sequence length, and the isolatednucleic acid molecules (e.g., oligonucleotides) are derived from thefirst or second halves of the molecules, or any of the four quarters.The human cMLCK cDNA, shown in SEQ ID NO: 1, can be used to illustratethis. The portion of the prototypical human cMLCK cDNA shown in SEQ IDNO: 1 is 1791 nucleotides in length and so can be hypothetically dividedinto about halves (nucleotides 1-895 and 896-1791) or about quarters(nucleotides 1-448, 449-895, 896-1343 and 1344-1791).

In one embodiment, nucleic acid molecules are selected that comprise atleast 10, 15, 20, 25, 30, 35, 40, 50 or 100 or more consecutivenucleotides of any of these or other portions of the human cMLCK cDNA,or of the 5′ or 3′ flanking regions. Thus, representative nucleic acidmolecules might include at least 10 consecutive nucleotides of theregion comprising nucleotides 1-448, 449-895, 896-1343 and 1344-1791 ofthe disclosed human cMLCK coding sequence.

EXAMPLE 2 Cloning of the cMLCK Genomic Gene

Some mutations in the cMLCK gene can lead to development or progressionof cardiac dysfunction (e.g., cardiomyopathy) are not included in thecDNA but rather are located in other regions of the cMLCK gene.Mutations located outside of the open reading frame that encodes thecMLCK protein are not likely to affect the functional activity of theprotein but rather are likely to result in altered levels of the proteinin the cell. In one embodiment, a mutation in the promoter region of thecMLCK gene prevents transcription of the gene and therefore leads to thecomplete absence of the cMLCK protein in the cell. Alternatively, amutation in the promoter region leads to unregulated or mis-regulatedexpression of cMLCK, including for instance overexpression ormislocalized or mis-timed expression.

Additionally, mutations within intron sequences in the genomic gene canalso prevent expression of the cMLCK protein. Following transcription ofa gene containing introns, the intron sequences are removed from the RNAmolecule in a process termed splicing prior to translation of the RNAmolecule which results in production of the encoded protein. When theRNA molecule is spliced to remove the introns, the cellular enzymes thatperform the splicing function recognize sequences around the intron/exonborder and in this manner recognize the appropriate splice sites. Ifthere is a mutation within the sequence of the intron close to thejunction of the intron with an exon, the enzymes may not recognize thejunction and can thus fail to remove the intron. If this occurs, theencoded protein can be defective. Thus, mutations inside the intronsequences within the cMLCK gene (termed “splice site mutations”) canalso lead to the development or progression of cardiac dysfunction.However, knowledge of the exon structure and intronic splice sitesequences of the cMLCK gene is required to define the molecular basis ofthese abnormalities. The provision herein of the cMLCK genomic structureand intron-exon boundaries (see Example 17) enables diagnosis of agenetic predisposition to cardiac dysfunction and cardiomyopathy basedon DNA analysis, and allows an analysis of all possible mutagenic eventsat the cMLCK locus.

With the sequences of the cMLCK cDNA and cMLCK gene in hand, primersderived from these sequences can be used in diagnostic tests (describedbelow) to determine the presence of mutations (including genomicamplifications or deletions) in any part of the genomic cMLCK gene of asubject, as well as 3′ and 5′ flanking sequences. Such primers can be,for example oligonucleotides including a fragment of sequence from thecMLCK gene (intron sequence, exon sequence or a sequence spanning anintron-exon boundary, or flanking region) and can include, for example,at least 10 consecutive nucleotides of the cMLCK cDNA or gene. It willbe appreciated that greater specificity can be achieved by using primersof greater lengths. Thus, in order to obtain enhanced specificity, theprimers used can comprise at least 10, 15, 17, 20, 23, 25, 30, 40 oreven 50 or 100 or more consecutive nucleotides of the cMLCK cDNA, geneor flanking region. Furthermore, with the provision of the cMLCK intronsequence information, the analysis of a large and as yet untapped sourceof patient material for mutations will now be possible using methodssuch as chemical cleavage of mismatches (Cotton et al., Proc. Natl.Acad. Sci. USA 85:4397-4401, 1985; Montandon et al., Nucleic Acids Res.9:3347-3358, 1989) and single-strand conformational polymorphismanalysis (Orita et al., Genomics 5:874-879, 1989).

Additional experiments can be performed to identify and characterizeregulatory elements flanking the cMLCK gene. These regulatory elementscan be characterized by standard techniques including deletion analyseswherein successive nucleotides of a putative regulatory region areremoved and the effect of the deletions are studied by either transientor long-term expression analyses experiments. The identification andcharacterization of regulatory elements flanking the genomic cMLCK genecan be made by functional experimentation (deletion analyses, etc.) inmammalian cells by either transient or long-term expression analyses.

Either the genomic clone or the cDNA or sequences derived from theseclones can be used in applications of this invention, including but notlimited to, studies of the expression of the cMLCK gene, studies of thefunction of the cMLCK protein, the generation of antibodies to the cMLCKprotein diagnosis and therapy of cMLCK amplified, deleted or mutatedpatients to prevent or treat the onset or progression of cardiacdysfunction or cardiomyopathy. Descriptions of applications of the useof cMLCK cDNA are therefore intended to comprehend the use of thegenomic cMLCK gene. It will also be apparent to one skilled in the artthat homologs of this gene can now be cloned from other species, such asthe rat, by standard cloning methods. Such homologs are useful in theproduction of animal models of cardiac dysfunction onset and diseaseprogression. In general, such orthologous cMLCK molecules will share atleast 50% sequence identity with the human cMLCK nucleic acid disclosedherein; more closely related orthologous sequences will share at least60%, at least 75%, at least 80%, at least 90%, at least 95%, or at least98% sequence identity with this sequence.

EXAMPLE 3 Nucleotide and Amino Acid Sequence Variants of cMLCK

With the provision of human cMLCK protein and corresponding nucleic acidsequences herein, the creation of variants of these sequences is nowenabled.

Variant cMLCK proteins include proteins that differ in amino acidsequence from the human cMLCK sequences disclosed but that share atleast 50% amino acid sequence homology with the provided human cMLCKprotein. Other variants will share at least 60%, at least 75%, at least80%, at least 90%, at least 95%, or at least 98% amino acid sequencehomology. Manipulation of the nucleotide sequence of cMLCK usingstandard procedures, including for instance site-directed mutagenesis orPCR, can be used to produce such variants. The simplest modificationsinvolve the substitution of one or more amino acids for amino acidshaving similar biochemical properties. These “conservativesubstitutions” are likely to have minimal impact on the activity of theresultant protein. Table 2 shows amino acids that may be substituted foran original amino acid in a protein, and which are regarded asconservative substitutions.

TABLE 2 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in enzymatic function or other protein featuresmay be obtained by selecting amino acid substitutions that are lessconservative than those listed in Table 2. Such changes include changingresidues that differ more significantly in their effect on maintainingpolypeptide backbone structure (e.g., sheet or helical conformation)near the substitution, charge or hydrophobicity of the molecule at thetarget site, or bulk of a specific side chain. The followingsubstitutions are generally expected to produce the greatest changes inprotein properties: (a) a hydrophilic residue (e.g., seryl or threonyl)is substituted for (or by) a hydrophobic residue (e.g., leucyl,isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline issubstituted for (or by) any other residue; (c) a residue having anelectropositive side chain (e.g., lysyl, arginyl, or histidyl) issubstituted for (or by) an electronegative residue (e.g., glutamyl oraspartyl); or (d) a residue having a bulky side chain (e.g.,phenylalanine) is substituted for (or by) one lacking a side chain(e.g., glycine).

Using these techniques, 800 samples from human subjects were screenedfor mutations in the cMLCK gene. Several mutations were identified, andare presented in Table 3.

One individual with MCV had a double point mutation, at cMLCK residues87 and 95. The cDNA encoding this mutant cMLCK was isolated, cloned intoa baculovirus vector, expressed in sF9 cells, and purified as describedin Example 4. In cMLCK activity assays similar to those described inExample 15, this mutant showed enhanced ability to incorporate phosphateinto RLC substrate, as manifested by an increased maximal velocity(about twice the maximal velocity of the unmutated enzyme).

Two true alleles were also identified. One, ala 89 glu, has an 8%prevalence in African-American populations. The other, pro 144 ala, hasabout 3% incidence in the general population.

TABLE 3 cMLCK Amino acid residue 87: GCG ala combined 87 and 95 mutationassociated mutated to: CTG val with MVCH; cMLCK with increased Vmax 95:GCA ala mutated to: GAA glu 89: GGC gly allele, est 8% incidence inAfrican Americans mutated to: GAC asp 144: CCT pro allele, est 3%incidence in general population mutated to: GCT ala

In a subsequence analysis, using these techniques, 300 African Americanpatients with hypertension were evaluated for the presence of the ala 89glu allele. Of this patient population, 25 individuals were heterozygousfor the ala 89 glu allele, while one individual was homozygous for theala 89 glu allele. The homozygous individual had left ventricularhypertrophy.

Variant cMLCK encoding sequences can be produced by standard DNAmutagenesis techniques, for example, M13 primer mutagenesis. Details ofthese techniques are provided in Sambrook et al. (In Molecular Cloning.A Laboratory Manual, CSHL, New York, 1989), Ch. 15. PCR-based and othermutagenesis techniques are also suitable. Details of some of thesetechniques are presented in Ausubel et al. (Short Protocols in MolecularBiology, 4th edition, Wiley, New York, 1999). By the use of suchtechniques, variants can be created which differ in minor ways from thehuman cMLCK sequences disclosed. DNA molecules and nucleotide sequencesthat are derivatives of those specifically disclosed herein, and whichdiffer from those disclosed by the deletion, addition, or substitutionof nucleotides while still encoding a protein that has at least 50%sequence identity with the cMLCK sequence disclosed (SEQ ID NO: 1), arecomprehended by this invention. Also comprehended are more closelyrelated nucleic acid molecules that share at least 60%, at least 75%, atleast 80%, at least 90%, at least 95%, or at least 98% nucleotidesequence homology with the disclosed cMLCK sequences. In their mostsimple form, such variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced.

Alternatively, the coding region can be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed human cMLCK protein sequences.For example, the 87th amino acid residue from the amino-terminus of thehuman cMLCK protein is alanine. The nucleotide codon triplet GCG encodesthis alanine residue. Because of the degeneracy of the genetic code,three other nucleotide codon triplets—GCT, GCC and GCA—also code foralanine. Thus, the nucleotide sequence of the human cMLCK ORF can bechanged at this position to any of these three alternative codonswithout affecting the amino acid composition or characteristics of theencoded protein. Based upon the degeneracy of the genetic code, variantDNA molecules can be derived from the cDNA and gene sequences disclosedherein using standard DNA mutagenesis techniques as described above, orby synthesis of DNA sequences. Thus, this invention also encompassesnucleic acid sequences which encode a cMLCK protein, but which vary fromthe disclosed nucleic acid sequences by virtue of the degeneracy of thegenetic code.

Variants of the cMLCK protein can also be defined in terms of theirsequence identity with the prototype cMLCK protein shown in SEQ ID NO:2. As described above, human cMLCK proteins share at least 50%, at least60%, at least 75%, at least 80%, at least 90%, at least 95%, or at least98% amino acid sequence identity with the human cMLCK protein disclosedherein (SEQ ID NO: 2). Nucleic acid sequences that encode such proteinscan readily be determined simply by applying the genetic code to theamino acid sequence of an cMLCK protein, and such nucleic acid moleculescan readily be produced by assembling oligonucleotides corresponding toportions of the sequence.

Nucleic acid molecules that are derived from the human cMLCK cDNAnucleic acid sequences disclosed include molecules that hybridize understringent conditions to the disclosed prototypical cMLCK nucleic acidmolecules, or fragments thereof. Specific non-limiting examples ofstringent conditions are hybridization at 65° C. in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg sheared salmon testes DNA, followed by15-30 minute sequential washes at 65° C. in 2×SSC, 0.5% SDS, followed by1×SSC, 0.5% SDS and finally 0.2×SSC, 0.5% SDS.

Low stringency hybridization conditions (to detect less closely relatedhomologs) are performed as described above but at 50° C. (bothhybridization and wash conditions); however, depending on the strengthof the detected signal, the wash steps can be terminated after the first2×SSC wash.

Human cMLCK nucleic acid encoding molecules (including SEQ ID NO: 1),and orthologs and homologs of these sequences can be incorporated intotransformation or expression vectors.

EXAMPLE 4 Expression of cMLCK Locus Polypeptides

With the provision of the human cMLCK cDNA, the expression andpurification of the cMLCK protein by standard laboratory techniques isnow enabled. In addition, proteins or polypeptides encoded by theantisense strand of the cMLCK cDNA can likewise be expressed. Afterexpression, the purified cMLCK locus protein or polypeptide can be usedfor functional analyses, antibody production, diagnostics, and patienttherapy. Furthermore, the DNA sequence of the cMLCK cDNA and itsantisense strand can be manipulated in studies to understand theexpression of the gene and the function of its product, as well as thefunction of the associated cMLCK locus. Mutant forms of the human cMLCKcan be isolated based upon information contained herein, and can bestudied in order to detect alteration in expression patterns in terms ofrelative quantities, tissue specificity and functional properties of theencoded mutant cMLCK protein. Partial or full-length cDNA sequences,which encode for the subject protein, can be ligated into bacterialexpression vectors. Methods for expressing large amounts of protein froma cloned gene introduced into Escherichia coli (E. coli) can be used forthe purification, localization and functional analysis of proteins. Forexample, fusion proteins consisting of amino terminal peptides encodedby a portion of the E. coli lacZ or trpE gene linked to cMLCK proteinscan be used to prepare polyclonal and monoclonal antibodies againstthese proteins (see below). Thereafter, these antibodies can be used topurify proteins by immunoaffinity chromatography, in diagnostic assaysto quantitate the levels of protein and to localize proteins in tissuesand individual cells by immunofluorescence.

Intact native protein can also be produced in E. coli in large amountsfor functional studies. Methods and plasmid vectors for producing fusionproteins and intact native proteins in bacteria are described inSambrook et al. (In Molecular Cloning: A Laboratory Manual, Ch. 17,CSHL, New York, 1989), and Ausubel et al. (Short Protocols in MolecularBiology, 4th edition, Chapter 16, Wiley, New York, 1999). Such fusionproteins can be made in large amounts, are easy to purify, and can beused to elicit antibody response. Native proteins can be produced inbacteria by placing a strong, regulated promoter and an efficientribosome-binding site upstream of the cloned gene. If low levels ofprotein are produced, additional steps can be taken to increase proteinproduction; if high levels of protein are produced, purification isrelatively easy. Suitable methods are presented in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and arewell known in the art. Often, proteins expressed at high levels arefound in insoluble inclusion bodies. Methods for extracting proteinsfrom these aggregates are described by Sambrook et al. (In MolecularCloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989) and Ausubelet al. (Short Protocols in Molecular Biology, 4th edition, Chapter 16,Wiley, New York, 1999). Vector systems suitable for the expression oflacZ fusion genes include the pUR series of vectors (Ruther andMuller-Hill, EMBO J. 2:1791, 1983), pEX1-3 (Stanley and Luzio, EMBO J.3:1429, 1984) and pMR100 (Gray et al., Proc. Natl. Acad. Sci. USA79:6598, 1982). Vectors suitable for the production of intact nativeproteins include pKC30 (Shimatake and Rosenberg, Nature 292:128, 1981),pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3 (Studiar andMoffatt, J. Mol. Biol. 189:113, 1986). cMLCK fusion proteins can beisolated from protein gels, lyophilized, ground into a powder and usedas an antigen. The DNA sequence can also be transferred from itsexisting context to other cloning vehicles, such as other plasmids,bacteriophages, cosmids, animal viruses and yeast artificial chromosomes(YACs) (Burke et al., Science 236:806-812, 1987). These vectors can thenbe introduced into a variety of hosts including somatic cells, andsimple or complex organisms, such as bacteria, fungi (Timberlake andMarshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasserand Fraley, Science 244:1293, 1989), and animals (Pursel et al., Science244:1281-1288, 1989), which cell or organisms are rendered transgenic bythe introduction of the heterologous cMLCK cDNA.

For expression in mammalian cells, the cDNA sequence can be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076,1981), and introduced into cells, such as monkey COS-1 cells (Gluzman,Cell 23:175-182, 1981), to achieve transient or long-term expression.The stable integration of the chimeric gene construct can be maintainedin mammalian cells by biochemical selection, such as neomycin (Southernand Berg, J. Mol. Appl. Genet. 1:327-341, 1982) and mycophenolic acid(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNAwith an intron and its own promoter) can be introduced into eukaryoticexpression vectors by conventional techniques. These vectors aredesigned to permit the transcription of the cDNA in eukaryotic cells byproviding regulatory sequences that initiate and enhance thetranscription of the cDNA and ensure its proper splicing andpolyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus andpolyadenylation and splicing signal from SV40 are readily available(Mulligan et al., Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gormanet al., Proc. Natl. Acad. Sci. USA 78:6777-6781, 1982). The level ofexpression of the cDNA can be manipulated with this type of vector,either by using promoters that have different activities (for example,the baculovirus pAC373 can express cDNAs at high levels in S. frugiperdacells (Summers and Smith, A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas Agricultural ExperimentStation Bulletin No. 1555, 1987; Ausubel et al., Chapter 16 in ShortProtocols in Molecular Biology, 1999) or by using vectors that containpromoters amenable to modulation, for example, theglucocorticoid-responsive promoter from the mouse mammary tumor virus(Lee et al., Nature 294:228, 1982). The expression of the cDNA can bemonitored in the recipient cells 24 to 72 hours after introduction(transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell. Biol. 1:486, 1981) orEpstein-Barr (Sugden et al., Mol. Cell. Biol. 5:410, 1985).Alternatively, one can also produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce thegene product on a continuous basis. One can also produce cell lines thathave amplified the number of copies of the vector (and therefore of thecDNA as well) to create cell lines that can produce high levels of thegene product (Alt et al., J. Biol. Chem. 253:1357, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. The vectors areintroduced into the recipient cells as pure DNA (transfection) by, forexample, precipitation with calcium phosphate (Graham and vander Eb,Virology 52:466, 1973) or strontium phosphate (Brash et al., Mol. Cell.Biol. 7:2013, 1987), electroporation (Neumann et al., EMBO J. 1:841,1982), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413,1987), DEAE dextran (McCuthan et al., J. Natl. Cancer Inst. 41:351,1968), microinjection (Mueller et al., Cell 15:579, 1978), protoplastfusion (Schafner, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980), orpellet guns (Klein et al., Nature 327:70, 1987). Alternatively, thecDNA, or fragments thereof, can be introduced by infection with virusvectors. Systems are developed that use, for example, retroviruses(Bernstein et al., Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al.,J. Virol. 57:267, 1986), or Herpes virus (Spaete et al., Cell 30:295,1982). cMLCK encoding sequences can also be delivered to target cells invitro via non-infectious systems, for instance liposomes.

These eukaryotic expression systems can be used for studies of cMLCKencoding nucleic acids and mutant forms of these molecules, the cMLCKprotein and mutant forms of this protein. Such uses include, forexample, the identification of regulatory elements located in the 5′region of the cMLCK gene on genomic clones that can be isolated fromhuman genomic DNA libraries using the information contained in thepresent invention. The eukaryotic expression systems can also be used tostudy the function of the normal complete protein, specific portions ofthe protein, or of naturally occurring or artificially produced mutantproteins.

Using the above techniques, the expression vectors containing the cMLCKgene sequence or cDNA, or fragments or variants or mutants thereof, canbe introduced into human cells, mammalian cells from other species ornon-mammalian cells as desired. The choice of cell is determined by thepurpose of the treatment. For example, monkey COS cells (Gluzman, Cell23:175-182, 1981) that produce high levels of the SV40 T antigen andpermit the replication of vectors containing the SV40 origin ofreplication can be used. Similarly, Chinese hamster ovary (CHO), mouseNIH 3T3 fibroblasts or human fibroblasts or lymphoblasts can be used.

The present invention thus encompasses recombinant vectors that compriseall or part of the cMLCK gene or cDNA sequences, or all or part of theantisense strand associated with the cMLCK-related locus, for expressionin a suitable host. The cMLCK DNA is operatively linked in the vector toan expression control sequence in the recombinant DNA molecule so thatthe cMLCK polypeptide can be expressed. The expression control sequencecan be selected from the group consisting of sequences that control theexpression of genes of prokaryotic or eukaryotic cells and their virusesand combinations thereof. The expression control sequence can bespecifically selected from the group consisting of the lac system, thetrp system, the tac system, the trc system, major operator and promoterregions of phage lambda, the control region of fd coat protein, theearly and late promoters of SV40, promoters derived from polyoma,adenovirus, retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

In one embodiment, the host cell, which is transfected with the vectoris selected from the group consisting of E. coli, Pseudomonas, Bacillussubtilis, Bacillus stearothermophilus or other bacilli; other bacteria;yeast; fungi; insect; mouse or other animal; or plant hosts; or humantissue cells.

It is appreciated that for mutant or variant cMLCK DNA sequences,similar systems are employed to express and produce the mutant product.In addition, fragments of the cMLCK protein are expressed essentially asdetailed above. Such fragments include individual cMLCK protein domainsor sub-domains, as well as shorter fragments such as peptides. cMLCKprotein fragments having therapeutic properties can be expressed in thismanner also.

It can be advantageous to express portions or fragments of the antisensestrand of the cMLCK locus (as delineated by SEQ ID NO: 1), or of regionsof the genome immediately upstream or especially immediately downstreamof this locus, but which overlap the disclosed sequences.

The full length human cMLCK cDNA was subcloned into pVL1393 Baculovirustransfer vector under the polyhedrin promoter with a FLAG® tag at 5′ endof MLCK. The baculovirus containing MLCK gene was then constructed withthe BaculoGold system (Pharmigen) from the transfer vector. The MLCKprotein was expressed by infecting the virus into the SF9 insect cellsand purified by anti-FLAG® affinity agarose resin (Sigma). The purifiedMLCK was dialyzed into a buffer contain 10 mM MOPS, 0.5 mM EGTA, 0.2 MNaCl, 1 mM dithiothreitol (DTT), and 10% glycerol with final pH 7.0.

EXAMPLE 5 Suppression of cMLCK Locus Expression

A reduction of cMLCK locus protein expression in a transgenic cell canbe obtained by introducing into cells an antisense construct based onthe cMLCK locus (SEQ ID NO: 1), including, for example, the reversecomplement of the cMLCK cDNA coding sequence, the cMLCK cDNA or genesequence or flanking regions thereof. For antisense suppression, anucleotide sequence from the cMLCK locus, e.g. all or a portion of thecMLCK cDNA or gene or the reverse complement thereof, is arranged inreverse orientation relative to the promoter sequence in thetransformation vector. Where the reverse complement of the reportedsequences is used to suppress expression of proteins from the cMLCKlocus, the sense strand of the disclosed cMLCK locus or cDNA is insertedinto the antisense construct. Other aspects of the vector can be chosenas discussed above (Example 4).

The introduced sequence need not be the full length human cMLCK cDNA orgene or reverse complement thereof, and need not be exactly homologousto the equivalent sequence found in the cell type to be transformed.Generally, however, where the introduced sequence is of shorter length,a higher degree of homology to the native cMLCK locus sequence is neededfor effective antisense suppression. In one embodiment, the introducedantisense sequence in the vector is at least 30 nucleotides in length,and improved antisense suppression is typically observed as the lengthof the antisense sequence increases. In another embodiment, the lengthof the antisense sequence in the vector is greater than 100 nucleotides.For suppression of the cMLCK gene itself, transcription of an antisenseconstruct results in the production of RNA molecules that are thereverse complement of mRNA molecules transcribed from the endogenouscMLCK gene in the cell. For suppression of protein expression from theopposite strand of the cMLCK locus, transcription of an antisenseconstruct results in the production of RNA molecules that are identicalto the mRNA molecules transcribed from the endogenous cMLCK gene,assuming the antisense construct was generated from sequence within thecMLCK gene rather than in a flanking region. Antisense molecules made totarget the sequence that is the reverse complement of the reported cMLCKlocus serve to suppress any abnormal expression of proteins or peptidesfrom the strand of the locus not encoding the cMLCK cDNA.

Although the exact mechanism by which antisense RNA molecules interferewith gene expression has not been elucidated, it is believed thatantisense RNA molecules bind to the endogenous mRNA molecules andthereby inhibit translation of the endogenous mRNA.

Suppression of endogenous cMLCK locus expression can also be achievedusing ribozymes. Ribozymes are synthetic RNA molecules that possesshighly specific endoribonuclease activity. The production and use ofribozymes are disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat.No. 5,543,508 to Haselhoff. The inclusion of ribozyme sequences withinantisense RNAs may be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

Finally, dominant negative mutant forms of the disclosed sequences canbe used to block endogenous cMLCK activity. Peptides derived from thecalmodulin-binding domain and neighboring residues are particularlylikely to have inhibitory effects. For example, a peptide comprising theC-terminal 46 residues of cMLCK: NNLAEKAKRC NRRLKSQILL KKYLMKRRWKKNFIAVSAAN RFKKISSSGA LMALGV (SEQ ID NO: 25)

includes a consensus calmodulin binding domain and putative MLCKautoinhibitory region, and is predicted to be a strong peptide inhibitorof cMLCK.

Suppression of cMLCK expression or expression of other proteins orpeptides encoded for by sequences within the cMLCK locus (including onthe reverse complement of the cMLCK cDNA) can be, for instance, used totreat cardiomyopathy and other forms of cardiac dysfunction caused byabnormalities in the cMLCK locus.

EXAMPLE 6 Production of Specific Binding Agents

Monoclonal or polyclonal antibodies can be produced to either the normalcMLCK protein or mutant forms of this protein, as well as to proteins orpeptides encoded for by the reverse complement of the disclosed cMLCKlocus sequences. Optimally, antibodies raised against these proteins orpeptides specifically detect the protein or peptide with which theantibodies are generated. That is, an antibody generated to the cMLCKprotein or a fragment thereof recognizes and binds the cMLCK protein anddoes not substantially recognize or bind to other proteins found inhuman cells. Such antibodies can be produced that are specific for thephosphorylated form of human cardiac RLC, as well as fragments andvariants of the phosphorylated form of human cardiac RLC.

The determination that an antibody specifically detects the cMLCKprotein or phosphorylated human cardiac RLC is made by any one of anumber of standard immunoassay methods; for instance, the Westernblotting technique (Ausubel et al., Short Protocols in MolecularBiology, 4th edition, Chapter 10, Wiley, New York, 1999). To determinethat a given antibody preparation (such as one produced in a mouse)specifically detects the cMLCK protein by Western blotting, totalcellular protein is extracted from human cells (for example,cardiomyocytes) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are then transferred to amembrane (for example, nitrocellulose) by Western blotting, and theantibody preparation is incubated with the membrane. After washing themembrane to remove non-specifically bound antibodies, the presence ofspecifically bound antibodies is detected by the use of an anti-mouseantibody conjugated to a marker, such as an enzyme. In one embodiment,the enzyme is alkaline phosphatase. Application of an alkalinephosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium results in the production of a dense blue compound byimmunolocalized alkaline phosphatase. Antibodies that specificallydetect the cMLCK protein will, by this technique, be shown to bind tothe cMLCK protein band (which will be localized at a position on the geldetermined by its molecular weight). Non-specific binding of theantibody to other proteins can occur and may be detectable as a weaksignal on the Western blot. The non-specific nature of this binding isrecognized by the weak signal obtained on the Western blot relative tothe strong primary signal arising from the specific antibody-cMLCKprotein binding.

Substantially pure cMLCK protein or protein fragment (peptide) suitablefor use as an immunogen can be isolated from the transfected ortransformed cells as described above. Concentration of protein orpeptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms per milliliter. Monoclonal or polyclonal antibody to theprotein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the cMLCK protein (or other proteinsor peptides derived from the cMLCK locus) of identified and isolated asdescribed can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-497, 1975) orderivative methods thereof. Briefly, a mouse is repetitively inoculatedwith a few micrograms of the selected protein over a period of a fewweeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess un-fused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall (Meth. Enzymol. 70:419-439, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein are prepared by immunizing suitable animals with theexpressed protein (Example 4), which can be unmodified or modified toenhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant. Also, hostanimals vary in response to site of inoculations and dose, with eitherinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al. (J. Clin.Endocrinol. Metab. 33:988-991, 1971).

Booster injections are given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al. (In Handbook of Experimental Immunology,Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration ofantibody is usually in the range of about 0.1 to 0.2 mg/ml of serum(about 12 μM). Affinity of the antisera for the antigen is determined bypreparing competitive binding curves, as described, for example, byFisher (Manual of Clinical Immunology, Ch. 42, 1980).

C. Antibodies Raised against Synthetic Peptides

A third approach to raising antibodies against the subject cMLCK locusencoded proteins or peptides is to use one or more synthetic peptidessynthesized on a commercially available peptide synthesizer based uponthe predicted amino acid sequence of the cMLCK locus encoded protein orpeptide.

By way of example only, polyclonal antibodies to specific peptideswithin cMLCK can be generated using well-known techniques, some of whichare described in Ausubel et al. (Short Protocols in Molecular Biology,4th edition, Chapter 11, Wiley, New York, 1999). Polyclonal antibodiesare generated by injecting these peptides into a suitable animal, suchas rabbits, chickens, or goats. The antibody preparations are used inimmunolocalization and protein quantification studies of the cMLCKprotein.

As a further example, polyclonal antibodies were raised against aphosphorylated peptide modeled after the human cardiac RLCphosphorylation site. A peptide with the sequence GANSNVF (SEQ IDNO:26), with the serine phosphorylated, was synthesized and injectedinto rabbit's using standard techniques as described above. Noantibodies capable of specific binding to human cardiac RLC wereobtained. Therefore, a concatemer duplicating the above sequence wassynthesized: GANSNVFGANSNVF (SEQ ID NO: 15), with two phosphoserines.This peptide was injected into rabbits, and yielded a highly specificpolyclonal antibody which recognized human phosphorylated cardiac RLC,but did not recognize unphosphorylated human cardiac RLC, orphosphorylated RLC from mouse or rabbit. The antibody was used asdescribed in examples 12 and 13.

D. Antibodies Raised by Injection of cMLCK Encoding Sequence

Antibodies can be raised against proteins and peptides of the cMLCKlocus by subcutaneous injection of a DNA vector that expresses thedesired protein or peptide, or a fragment thereof, into laboratoryanimals, such as mice. Delivery of the recombinant vector into theanimals can be achieved using a hand-held form of the Biolistic system(Sanford et al., Particulate Sci. Technol. 5:27-37, 1987) as describedby Tang et al. (Nature 356:152-154, 1992). Expression vectors suitablefor this purpose include those that express the cMLCK locus encodingsequence under the transcriptional control of either the human β-actinpromoter or the cytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample; or for immunolocalization of the cMLCK protein.

For administration to human patients, antibodies, e.g., cMLCK specificmonoclonal antibodies, can be humanized by methods known in the art.Antibodies with a desired binding specificity can be commerciallyhumanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, Calif.).

EXAMPLE 7 Nucleic Acid-Based Diagnosis

One particular application of the cMLCK locus sequence informationpresented herein, and of the cMLCK cDNA sequence, is in the area ofgenetic testing for predisposition to cardiac dysfunction orcardiomyopathy owing to cMLCK locus deletion, genomic amplification ormutation. The gene sequence of the cMLCK gene, including intron-exonboundaries and associated 5′ and 3′ flanking regions, is also useful insuch diagnostic methods. Individuals carrying mutations in the cMLCKlocus or gene, or having amplifications or heterozygous or homozygousdeletions of the cMLCK locus or gene, are detected at the DNA level withthe use of a variety of techniques. For such a diagnostic procedure, abiological sample of the subject, which biological sample containseither DNA or RNA derived from the subject, is assayed for a mutated,amplified or deleted cMLCK locus or gene. Suitable biological samplesinclude samples containing genomic DNA or RNA obtained from subject bodycells, such as those present in peripheral blood, urine, saliva, tissuebiopsy, surgical specimen, amniocentesis samples and autopsy material.The detection in the biological sample of a mutant cMLCK locus or gene,a mutant cMLCK RNA, or an amplified or homozygously or heterozygouslydeleted cMLCK locus or gene, is performed by one of a number ofmethodologies, for example, those described below.

A. Detection of Unknown Mutations:

Unknown mutations can be identified through polymerase chain reactionamplification of reverse transcribed RNA (RT-PCR) or DNA isolated frombreast or other tissue, followed by direct DNA sequence determination ofthe products; single-strand conformational polymorphism analysis (SSCP)(for instance, see Hongyo et al., Nucleic Acids Res. 21:3637-3642,1993); chemical cleavage (including HOT cleavage) (Bateman et al., Am.J. Med. Genet. 45:233-240, 1993; reviewed in Ellis et al., Hum. Mutat.11:345-353, 1998); denaturing gradient gel electrophoresis (DGGE),ligation amplification mismatch protection (LAMP); or enzymatic mutationscanning (Taylor and Deeble, Genet. Anal. 14:181-186, 1999), followed bydirect sequencing of amplicons with putative sequence variations.

B. Detection of Known Mutations:

The detection of specific known DNA mutations can be achieved by methodssuch as hybridization using allele specific oligonucleotides (ASOs)(Wallace et al., CSHL Symp. Quant. Biol. 51:257-261, 1986), direct DNAsequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995,1988), the use of restriction enzymes (Flavell et al., Cell 15:25, 1978;Geever et al., 1981), discrimination on the basis of electrophoreticmobility in gels with denaturing reagent (Myers and Maniatis, ColdSpring Harbor Symp. Quant. Biol. 51:275-284, 1986), RNase protection(Myers et al., Science 230:1242, 1985), chemical cleavage (Cotton etal., Proc. Natl. Acad. Sci. USA 85:4397-4401, 1985), and theligase-mediated detection procedure (Landegren et al., Science 241:1077,1988). Oligonucleotides specific to normal or mutant cMLCK sequences arechemically synthesized using commercially available machines. Theseoligonucleotides are then labeled radioactively with isotopes (such as³²P) or non-radioactively, with tags such as biotin (Ward and Langer etal., Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981), and hybridized toindividual DNA samples immobilized on membranes or other solid supportsby dot-blot or transfer from gels after electrophoresis. These specificsequences are visualized by methods such as autoradiography orfluorometric (Landegren et al., Science 242:229-237, 1989) orcalorimetric reactions (Gebeyehu et al., Nucleic Acids Res.15:4513-4534, 1987). Using an ASO specific for a normal allele, theabsence of hybridization indicates a mutation in the particular regionof the gene, or deleted cMLCK gene. In contrast, if an ASO specific fora mutant allele hybridizes to a clinical sample then that indicates thepresence of a mutation in the region defined by the ASO.

C. Detection of Genomic Amplification or Deletion:

Gene dosage (copy number) can be important in neoplasms; it is thereforeadvantageous to determine the number of copies of cMLCK locus nucleicacids in samples of tissue, e.g. cardiac tissue. It can also beadvantageous to determine the copy number of certain portions of thedisclosed nucleic acids, for instance about the 3′-terminal half or the3′-terminal third of the disclosed cMLCK cDNA (SEQ ID NO: 1), or of the5′ or especially 3′ region of the gene can also be determined. Probesgenerated from the disclosed encoding sequence of cMLCK (cMLCK probes orprimers), or the reverse complement of the cMLCK encoding sequence, canbe used to investigate and measure genomic dosage in the q23 region ofchromosome 17, and more particularly in the cMLCK gene.

In one embodiment, the cMLCK locus is divided into shorter regions andonly certain regions are probed for amplification. By way of example,the human cMLCK locus, cDNA, ORF, coding sequence and gene sequences isapportioned into about halves or quarters based on sequence length, andthe isolated nucleic acid molecules (e.g., oligonucleotides) is derivedfrom the first or second halves of the molecules, or any of the fourquarters. For example, the portion of the prototypical human cMLCK cDNAshown in SEQ ID NO: 1 is 1791 nucleotides in length and so can bedivided into about halves (e.g., from about nucleotides 1-885 and fromabout nucleotides 886-1791) or about quarters (1-447, 448-885, 886-1343and 1344-1791). The cDNA also could be divided into smaller regions,e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, withsimilar effect. Another mode of division is to select the 5′ (upstream)and/or 3′ downstream region associated with the cMLCK cDNA or cMLCKgene.

Appropriate techniques for measuring gene dosage are known in the art;see for instance, U.S. Pat. No. 5,569,753 (“Cancer Detection Probes”)and Pinkel et al. (Nat. Genet. 20:207-211, 1998) (“High ResolutionAnalysis of DNA Copy Number Variation using Comparative GenomicHybridization to Microarrays”).

Determination of gene copy number in cells of a patient-derived sampleusing other techniques is known in the art. For example, cMLCKamplification in muscle-derived cell lines as well as unculturedcardiomyocytes or other cells is carried out using bicolor FISHanalysis. By way of example, interphase FISH analysis of cardiomyocytesis carried out as previously described (Barlund et al., Genes Chromo.Cancer 20:372-376, 1997). The hybridizations are evaluated using a Zeissfluorescence microscope. Approximately 20 non-overlapping nuclei withintact morphology based on DAPI counterstain are scored to determine themean number of hybridization signals for each test and reference probe.

For tissue microarrays, the FISH is performed as described in Kononen etal., Nat. Med. 4:844-847, 1998. Briefly, consecutive sections of thearray are deparaffinized, dehydrated in ethanol, denatured at 74° C. for5 minutes in 70% formamide/2×SSC, and hybridized with test and referenceprobes. The specimens containing tight clusters of signals or >3-foldincrease in the number of test probe as compared to control areconsidered as amplified. Microarrays are constructed as described inWO9944063A2 and WO9944062A1.

In another embodiment, overexpression of the cMLCK gene is detected bymeasuring the cellular level of cMLCK-specific mRNA. mRNA can bemeasured using techniques well known in the art, including for instanceNorthern analysis, RT-PCR and mRNA in situ hybridization.

The nucleic acid-based diagnostic methods of this invention can bepredictive of susceptibility to cardiac dysfunction and/orcardiomyopathy.

EXAMPLE 8 Protein-Based Diagnosis

An alternative method of diagnosing cMLCK locus or gene amplification,deletion or mutation, as well as abnormal cMLCK expression, is toquantitate the level of cMLCK locus-associated protein (for instance,cMLCK protein) in the cells of an individual. This diagnostic tool isuseful for detecting reduced levels of the cMLCK locus-associatedprotein which result from, for example, mutations in the promoterregions of the cMLCK gene or mutations within the coding region of thegene which produced truncated, non-functional or unstable polypeptides,as well as from deletions of a portion of or the entire cMLCK gene.Alternatively, duplications of the cMLCK locus can be detected as anincrease in the expression level of one or more cMLCK locus-associatedproteins. Such an increase in protein expression can also be a result ofan up-regulating mutation in the promoter region or other regulatory orcoding sequence within the cMLCK locus or cMLCK gene. Localizationand/or coordinated cMLCK expression (temporally or spatially) can alsobe examined using well known techniques. The determination of reduced orincreased cMLCK locus-associated protein levels (e.g., cMLCK or proteinsor peptides expressed from the cMLCK locus for instance from the reversecomplement of the cMLCK cDNA or gene sequence), in comparison to suchexpression in a normal cell, would be an alternative or supplementalapproach to the direct determination of cMLCK locus deletion,amplification or mutation status by the methods outlined above andequivalents. The availability of antibodies specific to cMLCK locusprotein(s) will facilitate the detection and quantitation of cellularcMLCK locus protein(s) by one of a number of immunoassay methods whichare well known in the art and are presented in Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods ofconstructing such antibodies are discussed above, in Example 6, andantibodies are described in Example 12.

Any immunoassay format (e.g., ELISA, Western blot, or RIA assay) can beused to measure cMLCK locus polypeptide or protein levels; comparison isto wild-type (normal) cMLCK levels, and an increase in cMLCK polypeptideis indicative of an abnormal biological condition such as neoplasia.Immunohistochemical techniques can also be used for cMLCK polypeptide orprotein detection. For example, a tissue sample is obtained from asubject, and a section stained for the presence of cMLCK using a cMLCKspecific binding agent (e.g., anti-cMLCK antibody) and any standarddetection system (e.g., one which includes a secondary antibodyconjugated to horseradish peroxidase). General guidance regarding suchtechniques can be found in, e.g., Bancroft and Stevens (Theory andPractice of Histological Techniques, Churchill Livingstone, 1982) andAusubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

For the purposes of quantitating a cMLCK locus protein, a biologicalsample of the subject, which sample includes cellular proteins, is used.Such a biological sample can be obtained from body cells, particularlycardiomyocytes. Quantitation of cMLCK locus protein is achieved byimmunoassay and compared to levels of the protein found in healthycells. A significant (for example, about a 30% or greater) reduction inthe amount of cMLCK locus protein in the cells of a subject compared tothe amount of cMLCK protein found in normal human cells could be takenas an indication that the subject may have deletions or mutations in thecMLCK gene locus, whereas a significant (for example, about a 30% orgreater) increase would indicate that a duplication (amplification) mayhave occurred. Deletion, mutation and/or amplification of or within thecMLCK locus, and substantial under- or over-expression of one or morecMLCK locus protein(s), may indicate cardiac dysfunction or apredilection to cardiac dysfunction or cardiomyopathy.

EXAMPLE 9 cMLCK Knockout and Overexpression Transgenic Animals

Mutant organisms that under-express or over-express cMLCK or anothercMLCK locus associated protein are useful for research. Such mutantsallow insight into the physiological and/or pathological role of cMLCKin a healthy and/or pathological organism. These mutants are“genetically engineered,” meaning that information in the form ofnucleotides has been transferred into the mutant's genome at a location,or in a combination, in which it would not normally exist. Nucleotidestransferred in this way are said to be “non-native.” For example, anon-cMLCK promoter inserted upstream of a native cMLCK gene would benon-native. An extra copy of a cMLCK gene on a plasmid, transformed intoa cell, would be non-native.

Mutants can be, for example, produced from mammals, particularlynon-human mammals, such as mice, that either over-express cMLCK orunder-express cMLCK or another cMLCK locus associated protein, or thatdo not express cMLCK at all. Over-expression mutants are made byincreasing the number of cMLCK genes in the organism, or by introducingan cMLCK gene into the organism under the control of a constitutive orviral promoter such as the mouse mammary tumor virus (MMTV) promoter; amuscle-specific promoter such as the cardiac ELC promoter (Vermuri etal., PNAS 96: 1048-1053, 1999); or the metallothionein promoter. Mutantsthat under-express cMLCK can be made by using an inducible orrepressible promoter, or by deleting the cMLCK gene, or by destroying orlimiting the function of the cMLCK gene, for instance by disrupting thegene by transposon insertion.

Antisense genes can be engineered into the organism, under aconstitutive or inducible promoter, to decrease or prevent cMLCK locusexpression, as discussed above in Example 5.

A gene is “functionally deleted” when, for example, genetic engineeringhas been used to negate or reduce gene expression to negligible levels.When a mutant is referred to in this application as having the cMLCKgene altered or functionally deleted, this refers to the cMLCK gene andto any ortholog of this gene. When a mutant is referred to as having“more than the normal copy number” of a gene, this means that it hasmore than the usual number of genes found in the wild-type organism,e.g., in the diploid mouse or human.

A mutant mouse over-expressing cMLCK is made by constructing a plasmidhaving the cMLCK gene driven by a promoter, such as the mouse mammarytumor virus (MMTV) promoter or the cardiac ELC2 promoter. In oneembodiment, this plasmid is introduced into mouse oocytes bymicroinjection. The oocytes are implanted into pseudopregnant females,and the litters are assayed for insertion of the transgene. Multiplestrains containing the transgene are then available for study.

An inducible system can be created in which the subject expressionconstruct is driven by a promoter regulated by an agent that can be fedto the mouse, such as tetracycline. Such techniques are well known inthe art (see, e.g., Pinkert et al., Transgenic Animal Technology: ALaboratory Handbook, Academic Press, San Diego, 1994).

A mutant knockout animal (e.g., mouse) from which a cMLCK locus gene isdeleted is made by removing coding regions of the cMLCK gene fromembryonic stem cells. The methods of creating deletion mutations byusing a targeting vector have been described (Thomas and Capecchi, Cell51:503-512, 1987; Pinkert et al., supra).

EXAMPLE 10 Transfer of cMLCK Sequences

Approaches for combating cardiac dysfunction and/or cardiomyopathy insubjects are disclosed herein.

Retroviruses have been considered a preferred vector for experiments inthe transfer of nucleic acids in vivo, with a high efficiency ofinfection and stable integration and expression (Orkin et al., Prog.Med. Genet. 7:130-142, 1988). The full-length cMLCK locus or gene orcDNA can be cloned into a retroviral vector and driven from either itsendogenous promoter or from the retroviral LTR (long terminal repeat).Other viral transfection systems may also be used for this type ofapproach, including adenovirus, adeno-associated virus (AAV) (McLaughlinet al., J. Virol. 62:1963-1973, 1988), Vaccinia virus (Moss et al.,Annu. Rev. Immunol. 5:305-324, 1987), Bovine Papilloma virus (Rasmussenet al, Methods Enzymol. 139:642-654, 1987) or members of the herpesvirusgroup such as Epstein-Barr virus (Margolskee et al., Mol. Cell. Biol.8:2837-2847, 1988).

Recent developments in gene therapy techniques include the use ofRNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al.(Science 273:1386-1389, 1996). This technique allows for site-specificintegration of cloned sequences, thereby permitting accurately targetedgene replacement.

In addition to delivery of cMLCK to cells using viral vectors, it ispossible to use non-infectious methods of delivery. For instance,lipidic and liposome-mediated gene delivery has recently been usedsuccessfully for transfection with various genes (for reviews, seeTempleton and Lasic, Mol. Biotechnol. 11: 175-180, 1999; Lee and Huang,Crit. Rev. Ther. Drug Carrier Syst. 14:173-206; and Cooper, Semin.Oncol. 23:172-187, 1996). For instance, cationic liposomes have beenanalyzed for their ability to transfect monocytic leukemia cells, andshown to be a viable alternative to using viral vectors (de Lima et al.,Mol. Membr. Biol. 16:103-109, 1999). Such cationic liposomes can also betargeted to specific cells through the inclusion of, for instance,monoclonal antibodies or other appropriate targeting ligands (Kao etal., Cancer Gene Ther. 3:250-256, 1996).

To reduce the level of cMLCK expression, gene therapy can be carried outusing antisense or other suppressive constructs, the construction ofwhich is discussed above (Example 5).

EXAMPLE 11 Diagnostic Kits

Kits are provided herein which contain the necessary reagents fordetermining gene copy number (genomic amplification), such as probes orprimers specific for the cMLCK gene, as well as written instructions.The instructions can provide calibration curves or charts to comparewith the determined (e.g., experimentally measured) values. Kits arealso provided to determine elevated expression of mRNA (i.e., containingprobes) or cMLCK locus-associated protein (i.e., containing antibodiesor other cMLCK-protein specific binding agents).

A. Kits For Detection of cMLCK Genomic Amplification

The nucleotide sequences disclosed herein, and fragments thereof, can besupplied in the form of a kit for use in detection of cMLCK locusgenomic amplification and/or diagnosis of cardiac dysfunction orcardiomyopathy. In such a kit, an appropriate amount of one or more ofthe cMLCK-specific oligonucleotide primers is provided in one or morecontainers. In one embodiment, the oligonucleotide primers are providedsuspended in an aqueous solution or as a freeze-dried or lyophilizedpowder. The container(s) in which the oligonucleotide(s) are suppliedcan be any conventional container that is capable of holding thesupplied form, for instance, microfuge tubes, ampoules, or bottles. Insome applications, pairs of primers are provided in pre-measured singleuse amounts in individual, typically disposable, tubes or equivalentcontainers. With such an arrangement, the sample to be tested for thepresence of cMLCK locus genomic amplification is added to the individualtubes and in vitro amplification carried out directly.

The amount of each oligonucleotide primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several PCR in vitroamplification reactions. Those of ordinary skill in the art know theamount of oligonucleotide primer that is appropriate for use in a singleamplification reaction. General guidelines may for instance be found inInnis et al. (PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (InMolecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y., 1989),and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1998). Alternative amplification methods mayalso be used (such as strand displacement amplification—U.S. Pat. Nos.5,744,311, 5,648,211, and 5,631,147).

A kit can include more than two primers, in order to facilitate the PCRin vitro amplification of cMLCK locus genomic sequences, for instancethe cMLCK gene or the 5′ or 3′ flanking region thereof.

In some embodiments of the current invention, the kits also include thereagents necessary to carry out PCR in vitro amplification reactions,including, for instance, DNA sample preparation reagents, appropriatebuffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), anddeoxyribonucleotides (dNTPs). Written instructions can also be included.

In another embodiment, kits include either labeled or unlabeledoligonucleotide probes for use in detection of the in vitro amplifiedcMLCK locus sequences. The appropriate sequences for such a probe is anysequence that falls between the annealing sites of the two providedoligonucleotide primers, such that the sequence the probe iscomplementary to is amplified during the PCR reaction.

One or more control sequences for use in the PCR reactions can also beprovided in the kit. The design of appropriate positive controlsequences is well known to one of ordinary skill in the appropriate art.

B. Kits For Detection of mRNA Overexpression

Kits similar to those disclosed above for the detection of cMLCK locusgenomic amplification can be used to detect cMLCK locus-associated mRNAoverexpression. Such kits include an appropriate amount of one or moreof the oligonucleotide primers for use in reverse transcription PCRreactions, similarly to those provided above, with art-obviousmodifications for use with RNA.

In some embodiments of the current invention, kits for detection ofcMLCK mRNA overexpression include the reagents necessary to carry outRT-PCR in vitro amplification reactions, including, for instance, RNAsample preparation reagents (including e.g., an RNAse inhibitor),appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesiumchloride), and deoxyribonucleotides (dNTPs). Written instructions canalso be included.

Kits in addition can include either labeled or unlabeled oligonucleotideprobes for use in detection of the in vitro amplified target sequences.The appropriate sequences for such a probe is any sequence that fallsbetween the annealing sites of the two provided oligonucleotide primers,such that the sequence the probe is complementary to is amplified duringthe PCR reaction.

In another embodiment, the kit includes one or more control sequencesfor use in the RT-PCR reactions. The design of appropriate positivecontrol sequences is well known to one of ordinary skill in theappropriate art.

Alternatively, kits can be provided with the necessary reagents to carryout quantitative or semi-quantitative Northern analysis of cMLCK locusmRNA. Such kits include, for example, at least one cMLCK-specificoligonucleotide for use as a probe. This oligonucleotide can be labeledin any conventional way, including with a selected radioactive isotope,enzyme substrate, co-factor, ligand, chemiluminescent or fluorescentagent, hapten, or enzyme label.

C. Kits For Detection of cMLCK Locus Protein or Peptide Overexpression

Kits for the detection of cMLCK locus-associated protein overexpression,for instance cMLCK protein overexpression, are also encompassed in thecurrent invention. Such kits include at least one target (e.g., cMLCK)protein specific binding agent (e.g., a polyclonal or monoclonalantibody or antibody fragment). In one embodiment, the kit includes atleast one control. The cMLCK protein specific binding agent and controlare contained in separate containers. Optionally, the kits can alsoinclude a means for detecting cMLCK:agent complexes, for instance theagent can be detectably labeled. If the detectable agent is not labeled,it can be detected by second antibodies or protein A for example whichcan also be provided in one or more separate containers.

Additional components in a kit includes instructions for carrying outthe assay. Instructions will allow the tester to determine whether cMLCKexpression levels are elevated. Optionally, reaction vessels andauxiliary reagents such as chromogens, buffers, enzymes, etc. can alsobe included in the kits.

EXAMPLE 12 Specific Antibodies Reacting with Phosphorylated Human andMouse RLC

To study phosphorylation of RLC by cardiac MLCK, a polyclonal antibodywas raised against peptides containing the RLC serine residue which isphosphorylated by MLCK. Peptides were synthesized containing this serineresidue in its phosphorylated state, with the bilateral flanking 3residues on either side. Both the mouse and human sequences weresynthesized. These were injected into rabbits as described in Example 6.

After an initial attempt to raise a useable antibody failed, inoculationof a rabbit with a concatamer of 2 such peptides (SEQ ID NO: 15)produced surprisingly specific antibodies. Two polyclonal antibodieswere made: one against human and another against murine sequence.

On Western blot, both anti-human RLC-P and anti-mouse RLC-P polyclonalantibodies detected only the phosphorylated human cardiac RLC. Theantibody against human peptide sequence was specific for humanphosphorylated cardiac RLC, while the antibody against murine sequencereacted against rabbit, murine and human phosphorylated cardiac RLC.

Cryosections of fresh human skeletal muscle tissue were prepared asdescribed in Ausubel et al., Chapter 14 of Short Protocols in MolecularBiology, 1999. Immunohistochemistry was performed using fluorescentlylabeled anti-rabbit secondary antibodies as described in Ausubel et al.

When the antibody against the phosphorylated human RLC was used as theprimary antibody, a mixture of distinct myofibers with and without RLCphosphorylation was observed. The pattern of staining did not resemblemotor units. Co-staining with antibodies to fast myosin showed that bothfast and slow myofibers contained RLC-P detected with this antibody.

In order to confirm this finding, the antibody raised against the murinephosphorylated RLC peptide was used for immunohistochemical analysis ofa variety of mouse and rabbit skeletal muscle cryosections. In eachcase, a patchy pattern of phosphorylated and non-phosphorylated fiberssimilar to the human sample was observed. In order to confirm theheterogeneous pattern RLC phosphorylation in skeletal muscle, individualmuscle fibers from rabbit psoas muscle were evaluated on a 10% glycerolgel that resolves phosphorylated RLC from non-phosphorylated fibers.Consistent with the immunohistochemical findings, the gel analysisshowed fibers with and without detectable RLC phosphorylation.

EXAMPLE 13 Distribution of Phosphorylated RLC and cMLCK in the Heart

In order to study the pattern of phosphorylated RLC in mouse heart, theantibody against the murine phosphorylated RLC described in example 12was used for immunohistochemical analysis of cryosections from murinehearts. In all cases, the maximal staining occurred at the apex andcontrasted with significantly less staining in the mid-region of theventricles and the papillary muscles. An intermediate intensity ofstaining was observed at the base. In addition, a gradient of expression(from greatest to least) was observed extending inward from epicardiumto endocardium. Technical artifact was ruled out using other antibodiesthat showed uniform staining throughout the heart. Western blot analysiswith tissue from apex, epicardium, endocardium, and papillary muscleprobed with the same antibody confirmed the non-uniform pattern ofexpression that was observed in immunohistochemical analysis.

In order to study the distribution of the MLCK cloned from human heart,a polyclonal antibody to the entire human protein was produced. The fulllength cDNA encoding the human MLCK protein was inserted into abaculoviral vector that was transfected with helper virus to produce therecombinant baculovirus which in turn was used to infect sF9 cells (seeExample 4 for details of expression and purification). A FLAG® tag(KODAK®), placed at the amino-terminal end of the MLCK permittedaffinity column purification directly from the SF9 cell homogenate.Rabbits were innoculated with this affinity purified protein and boostedat 2, 4, and 6 weeks. The 8 and 10 week serum was affinity purifiedusing affinity chromatography with full-length cMLCK. The serum from onerabbit detected the expressed human MLCK on Western blot although it wasless useful for fluorescent in situ detection of MLCK in fresh-frozenmouse heart.

Western blot analysis was performed on tissue from mouse and rabbithearts that were fresh frozen in super-cooled isopentane. Five micronsections from apex, epicardium, endocardium and inter-ventricular septumwere collected and pooled from multiple hearts. MLCK was extractedseparately from these regions and analyzed by Western blot. A matchedWestern blot performed on extracted myosin from similarly obtainedtissue was probed with the antibody to phosphorylated murine RLC. TheWestern blot analysis of both murine and rabbit heart tissue showed aregional distribution of the MLCK that matched the distribution ofphosphorylated RLC as detected by both fluorescent in situ studies andwestern blot analysis.

The non-uniform but reproducible staining pattern is inversely relatedto the pattern of hypertrophy in humans with mid-cavitary ventricularhypertrophy (MCV), and in mice expressing a human ELC mutant proteinassociated with MCV (Met149Val). Specifically, anti-MLC-Pimmunohistochemistry revealed strong staining of the apex, intermediatestaining at the base, and light staining of the mid-ventricularmyocardium and papillary muscle. Anti-cMLCK Western blot revealed thesame pattern.

It has previously been shown that transgenic mice expressing a humanmutant ELC (Met 149Val) faithfully reproduced the MCV phenotype ofpatients from whom the mutant ELC was derived. By comparing the patternof cardiac hypertrophy in M149V mice with the pattern of RLCphosphorylation in the normal mouse heart, it is observed that thenormally hypo-phosphorylated portion of the heart is the very regionthat hypertrophies in mice expressing the mutant ELC throughout theheart. In other words, the mid-ventricular areas of lightimmunohistochemical staining correspond exactly to the pattern of MCVobserved in humans and transgenic mice expressing mutant ELC.

EXAMPLE 14 Muscle Mechanics

The effects of mutations in human cMLCK can be investigated by examiningmyocardial mechanics in right ventricular papillary muscles obtainedfrom transgenic mice. Mice can be constructed expressing a human cMLCK,and mutant human cMLCK, or mutant human RLC (such as ala 13 thr and glu22 lys, as described in Poetter et al., Nature Genetics 13: 63-69,1996). Methods for examining myocardial mechanics of normal and mutantmice may be found in Vemuri et al., PNAS 96: 1048-1053, 1999, which isherein incorporated by reference in its entirety. These methods arereviewed in this Example.

Mice are killed, and the hearts are rapidly excised. The rightventricles are opened, and the papillary muscles are excised and pinnedinto a dish containing a high EGTA permeabilizing solution (Eastwood etal., Tissue Cell 11, 553-566, 1979) maintained at 2° C. After 4 hours,the solution is replaced by one containing 50% glycerol and is kept at20° C.

Measurement Apparatus. Each muscle strip examined is mounted between asilicon strain gauge force transducer and a servo motor in atemperature-controlled chamber at 20° C., with a low calciumconcentration (relaxing) solution. The system has a force transducerelement (Akers, Horten, Norway) with custom mounting, which has anatural resonance at 5.6 kHz but, with damping, has a flat frequencyresponse up to 3 kHz. The frequency response of the servo motor (6350;Cambridge Technology, Cambridge, Mass.) is limited by a resonance at1300 Hz. Control of muscle length is performed by using custom softwarewritten in the inventor's laboratory. The software controls aprogrammable filter (9002; Frequency Devices, Haverhill, Mass.) and adigital oscilloscope (model 54600A; Hewlett-Packard). Data consisting offorce and motor position (hence muscle length) are sampled by using anA/D board (DT2828; Data Translation, Marlboro, Mass.) with 12 bits ofresolution at a frequency of 5 kHz for quick stretch experiments. Fordynamic stiffness measurements, the sampling frequency varies from 16kHz at the high driving frequencies down to 40 Hz at a driving frequencyof 0.02 Hz. The programmable filter (8 pole Bessel with linear phase) isset to low pass filter at a frequency ⅛ (at high frequencies) to 1/64(at lower frequencies) of the data acquisition rate to avoid aliasing.Data are collected over 256 sinusoidal cycles at 500 Hz and over 1 cycleat 0.02 Hz, saved on an 80486 computer and are analyzed off-line.

Muscle Protocol. The muscle fibers are mounted and stretched in relaxingsolution to 110% of slack length. Muscle dimensions are measuredoptically under a dissecting microscope. Under computer control, theservo motor is driven sinusoidally at 55 selected frequencies from 500Hz to 0.02 Hz. The length changes chosen are 0.1% muscle length, butamplitudes between 0.05 and 0.5% should give similar results. Thesinusoidal amplitude of 0.1% is as small as technically feasible tominimize the nonlinearities of viscoelastic tissues. The signalsrepresenting motor position (muscle fiber length) and force aredigitized and recorded to computer disk. Then, the bathing solution ischanged briefly to a “preactivating solution” similar to the relaxingsolution but with 20 mM 1,6-hexamethylenediamine-N,N,N′,N′-tetraaceticacid replacing the EGTA. It then is changed to a high calcium solutionof the following composition, in mM: Na 51, K 86, Cl 13, creatinephosphate 20, EGTA 25, N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonicacid 100, MgATP 5, reduced glutathione 10, leupeptin 0.1, and sufficientCaEGTA to obtain a pCa of 4.3. After steady isometric force is reached,the muscle is step-wise stretched to 1% of its initial length, and theforce signal is recorded for 4 s. Then, the motor again is driven ateach of 55 discrete frequencies between 500 Hz and 0.02 Hz, and themotor position and force signals are recorded. The bathing solution isreplaced with an identical solution without any ATP or creatinephosphate with a flow-through wash of >5× the chamber volume. Thedynamic stiffness again is determined in this rigor solution.

Data analysis can include determining the isometric force before anystretches, displaying the force levels during the 1% step-wise stretch,and measuring the time-to-peak of the delayed force, the amplitude ofthe delayed force, and the force amplitude for 4 s of stretch. Initialisometric force is normalized to cross-sectional area to give stress(kN/m²). To compare the quick stretch experimental data, forces duringthese stretches are normalized to initial isometric prestretch force.During the experiments in which the fiber bundles were sinusoidallylengthened and shortened, the signals representing the changing fiberlength and the force were collected for each of the discretefrequencies.

The fast Fourier transform can be used on the length signal to determinethe driving frequency of the length oscillation, and its amplitude andphase. At this driving frequency, the fast Fourier transform of theforce signal is computed to determine the force oscillation amplitudeand phase. The response of the fibers is computed as the ratio of theforce amplitude to length amplitude (modulus, or stiffness) and theforce phase minus the length phase. The impedance at each frequencyconsists of both the magnitude ratio and phase. These transforms arecomputed at each frequency for each condition (relaxed, activated, andrigor). Thus, correction for the fiber response to seriesviscoelasticities (measured as end-compliance) is accomplished throughanalysis of rigor fibers preloaded to the tension of the activatedfiber. Parallel viscoelasticities are determined in fibers under relaxedconditions and are subtracted from the activated response.

Effect of RLC Phosphorylation in Skinned Muscle Fibers

The effect of regulatory light chain phosphorylation on stretchactivation was investigated in chemically skinned muscle fibers fromrabbit. This experimental model is well established in the art, and themodel with various modifications is described in detail in U.S. Pat. No.5,446,186; Davis, Advances in Experimental Medicine & Biology 453:343-51, 1998; Rapp et al., Journal of Muscle Research & Cell Motility17: 617-29, 1996; Davis et al., Biophysical Journal. 68: 2032-40, 1995;Davis et al., PNAS 92: 10482-10486, 1995; and Davis et al., BiophysicalJournal 65: 1886-1898, 1993; all of which are herein incorporated byreference.

The human cMLCK was expressed in sF9 cells and purified as described inExample 4. The purified cMLCK protein was activated by combining insolution with calcium-calmodulin, and used to phosphorylate myosin RLCsin the skinned muscle preparation. Control preparations were treatedwith solution without cMLCK.

These fibers were subjected to large stretches (0.4-0.8% muscle lengths)before and after RLC phosphorylation. Prior to stretch there is adramatic increase in isometric tension produced by the RLCphosphorylated fibers (RLCP) compared to the same fiber before RLCphosphorylation. On average there is a 2.5 fold increase in isometricfiber tension, from a baseline of 22% maximal tension to 56% of themaximal value. Consequently the tension increases from stretch and theensuing tension transient is noticeably greater in the RLCP containingfibers than in the non-phosphorylated RLC fibers. However, the relativetotal excursion (the sum of the fall and subsequent delayed rise intension) that occurs following the initial tension increase isproportionally greater in the RLC (17%) vs. RLCP (10%). The increasedtotal excursion represents a 7% larger stretch-activation response inthe fibers with the non-phosphorylated RLC. Relaxation kinetics studies,that impose small step-stretches on these fibers, have shown that thestretch-activation response (Huxley-Simmons phase 3) isdisproportionately increased in fibers without phosphorylated RLC(manuscript submitted). Thus, in small as well as large stretch studies,the increased tension produced by RLCP is associated with a reciprocaldrop in the stretch-activation response while the converse effect occursin non-phosphorylated fibers. These mechanical differences, associatedwith the gradient of RLC phosphorylation across the ventricular wall,support the complex pattern of cardiac torsion.

EXAMPLE 15 Methods of Screening for Compounds that Modulate cMLCKActivity

The reagents provided in this disclosure form the basis of a variety ofassays that can be used to identifying compounds that modulate cMLCKactivity. Such modulatory compounds may be, for example,pharmaceuticals, peptides, or antibodies, and may increase or decreasecMLCK activity.

In Vitro Assays for Myosin Light Chain Kinase Biological Activity

For example, a kinase activity assay is run in the presence and absenceof the test compound, and the impact of the compound on cMLCK's abilityto phosphorylate a suitable substrate is determined. Such assays forMLCK kinase biological activity are well-known in the art; see, forexample, U.S. Pat. No. 5,906,819; Ausubel et al., Short Protocols inMolecular Biology, 4th edition at p. 17-22. To assess the ability of aputative cMLCK modulatory compound to inhibit or activate cMLCK, aseries of kinase activity assays are carried out in the presence ofvarying concentrations of the putative modulatory compound (includingzero concentration), and the extent of phosphate incorporation intosubstrate is determined for each assay.

For example, the kinase reaction for myosin light chain kinase iscarried out in 50 μl of reaction mixture (50 mM Tris/HCl at pH 7.5, 1 mMMg_(Cl2), 85 mM KCl, 500 mM ATP, purified cMLCK (typically from 0.1 to100 ng of protein depending on reaction conditions), and myosin lightchain, a myosin light chain fragment or variant, or a synthetic peptidesubstrate capable of being phosphorylated by cMLCK and specificallyrecognized by an anti-human RLC-P antibody when phosphorylated. Anexample of a suitable synthetic peptide substrate, based on the sequencesurrounding the phosphorylatable serine of human cardiac MLC, isGGANSNVFSMFEQT (SEQ ID NO: 16). Reactions is initiated by addition ofenzyme or substrate. Incubation period is determined empirically basedon a variety of factors including enzyme and substrate concentration andincubation temperature. Commonly used conditions are about 10 minutes ofincubation at about 30° C. Reactions are carried out with or without 0.1mM Ca_(Cl2) and 10 μg/ml calmodulin. Duplicate reactions are carried outwith or without the compound being tested. Reactions are stopped byadding a calcium chelator such as 1 mM EGTA, and/or addition of sodiumdodecyl sulfate as described in U.S. Pat. No. 5,906,819. Once thereaction is stopped, the extent of substrate phosphorylation in eachreaction is determined.

Another suitable example protocol is described in Ausubel et al., ShortProtocols in Molecular Biology, 4th Edition at p. 17-22, and is similarto the protocol in U.S. Pat. No. 5,906,819. The following modificationsare made to adapt the assay for determination of myosin light chainkinase biological activity: the synthetic peptide substrate described inthe Ausubel et al. Protocols is replaced by a like amount of myosinlight chain, a myosin light chain fragment or variant, or a syntheticpeptide substrate capable of being phosphorylated by cMLCK andspecifically recognized by an anti-human RLC-P antibody whenphosphorylated. The calmodulin-dependent kinase used is cMLCK. After thereaction is stopped, the mixture is spotted onto phosphocellulose P81 ifa short peptide substrate; or nitrocellulose, PVDF, and the like if thesubstrate is a larger polypeptide.

A third suitable protocol is as follows. This protocol directly detectsincorporation of phosphate into substrate. The phosphorylation of myosinregulatory light chain is performed in the buffer containing 50 mM MOPS,pH 7.2, 10 mM MgAcetate, 1 mM DTT, 600 μM CaCl2, 2×10⁻⁷ M calmodulin,500 μM ATP with ³²P labeled gamma ATP, (30 ci/mMole) at 25° C. Otherforms of gamma-labeled ATP or GTP are also suitable. The final MLCKconcentration is 2.8 ng/100 μl. The reaction mixture with myosinregulatory light protein without MLCK is preincubated at 25° C. for 10minutes before MLCK is added. The assay is started by adding MLCK to theprecondition mixture and reaction time is recorded. The phosphorylationis stopped by spotting 20 μl of the reaction mixture each time toWhatman circle filter paper (Grade 3) and dropping the filter paper intocold (4° C.) stop solution with 10% TCA, 8% Napyrophosphate. The filterpaper is then washed three times in the wash solution containing 10%TCA, 2% Napyrophosphate. The washed filter paper is rinsed once with100% alcohol and three times with ether and air dried. The amount ofphosphorylated myosin regulatory light chain protein on the filter paperwith labeled ³²P ATP can be detected in the scintillation counter.

This protocol has the advantage of providing an alternate approach toquantification of cMLCK activity, one that provides an alternative toimmunoassay and does not require specific binding agents such as aphosphospecific antibody. By quantifying the amount of phosphateretained by the filter, the amount of phosphate incorporated into theRLC substrate is readily calculated. Since the enzyme and substrateconcentration, ATP concentration, ³²P specific activity and reactionconditions are known, steady-state kinetic parameters such as Vmax(enzyme half-maximal velocity) and Km (substrate concentration at whichenzyme velocity is half maximal) are readily calculated once the amountof phosphate retained by each filter is known. See, for example, Chapter8 in Stryer, Biochemistry 3rd Ed., W.H. Freeman & Co., 1988; Dixon andWebb, Enzymes 3rd Ed., Longmans 1979. The impact of a putativeinhibitory or activating compound on the enzyme's maximal velocity, Km,affinity for substrate, and affinity for ATP are also readily determinedusing the approaches and calculations described in Chapter 8 of Stryerand Dixon and Webb. In this way, a compound can be identified as aninhibitor or activator of cMLCK, and an initial assessment of itsrelative potency can be determined.

Determining Extent of Substrate Phosphorylation by Immunoassay

Determination of the extent of substrate phosphorylation can also bemade by any suitable immunoassay, such as those described in Harlow andLane, Antibodies: A Laboratory Manual, or Ausubel et al., CurrentProtocols in Molecular Biology, 1998. These include immunoblotting,immunoprecipitation, ELISA, radioimmunoassay, and immuno-affinity. Forexample, proteins in the cMLCK assay mixture can be immobilized on anysuitable substrate (for example, on nitrocellulose membranes;immobilization in a 6, 12, 24, or 96 well plate, etc.) and the amount ofphosphorylated substrate detected.

In one embodiment, phosphorylated substrate are detected using aspecific binding agent such as an antibody which specifically detects anRLC, or fragment or variant thereof, after MLCK-mediatedphosphorylation. For example, the polyclonal phosphospecific antibodydescribed in Example 6 is a suitable choice. Those skilled in the artrecognize that the substrate can be varied, and a broad range of otherantibodies can be used. For example, rabbit skeletal muscle RLC can be asuitable substrate for cMLCK, and antibodies could be raised thatspecifically detect the rabbit RLC phosphoserine residue. Monoclonalantibodies are also suitable.

The phosphospecific binding agent can be labeled in a variety of ways,as described in various references readily available to practitioners inthe art (see, for example, Harlow and Lane, Antibodies: A LaboratoryManual; Ausubel et al., Current Protocols in Molecular Biology, 1998).For example, the enzyme is linked to a fluorescent label, or linked toan agent such as digoxigenin or biotin that is readily recognized by asecondary binding agent. Commonly, the phosphospecific binding agent isused as a primary binding agent.

Following binding of the phosphospecific binding agent and appropriatewashing, a secondary binding agent capable of binding to thephosphospecific binding agent is used. An example is anti-mouse Fcantibodies, when the phosphospecific binding agent is a mouse monoclonalantibody. The secondary antibody can be conjugated to an enzyme such asalkaline phosphatase or horseradish peroxidase, a fluorescent label, orthe like, and detected by spectroscopy, autofluorography,chemiluminescence, etc. as indicated. As another alternative, thesecondary antibody can be, for example, an anti-digoxigenin, anti-biotinantibody, anti-DNP antibody, or anti-fluorescein isothiocyanate antibodyconjugated to an appropriate label. The choice of secondary antibodydepends on the nature of the primary specific binding agent. Forexample, if the phosphospecific binding agent is a polyclonal antibodyconjugated to digoxigenin, an anti-digoxigenin antibody conjugated toalkaline phosphatase or fluorescein isothiocyanate would be anappropriate choice for secondary antibody.

After completing binding of specific binding agents and washing, theamount of phosphospecific antibody bound is determined.Enzyme-conjugated antibodies can be detected by visual inspection ofcolor development, spectroscopy, and chemiluminescence, depending on thereagents used. For example, a bound alkaline phosphatase-conjugatedantibody can be detected by chemiluminescence after incubation withELISA-LIGHT™ (Applied Biosciences) reagent according to themanufacturer's instructions.

Usually, conjugated secondary antibodies and detection kits are obtainedcommercially from suppliers such as Dako, Applied Biosciences, andOxford Biomedical Research. These kits are supplied with detailedinstructions for use in particular kinds of immunoassays.

Another well-known approach is to immobilize a binding agent capable ofbinding phosphorylated cMLCK substrate, and contacting the immobilizedbinding agent with the substrate in solution at the completion of theassay for myosin light chain kinase biological activity.

For example, the antibody-sandwich ELISA described at p. 11-8 and 11-9of Ausubel et al., Short Protocols in Molecular Biology could besuitably adapted to determining the amount of phosphate incorporatedinto substrate. A phosphospecific antibody such as that described inExample 6 can be used as the “capture antibody.” The capture antibody isbound to the coat wells of an Immulon plate as described in Ausubel etal., and contacted with a solution containing RLC substrate.Phosphorylated RLC is bound, whereas unphosphorylated RLC is not. Asecondary antibody in this assay can be a second specific anti-RLCantibody (specifically binding to an epitope other than thephosphorylated serine). Alternatively, the RLC substrate can have anepitope tag such as a FLAG® tag, and the secondary antibody ananti-epitope tag antibody. The secondary antibody can be linked to anysuitable detectable label.

Those skilled in the art recognize that when a phosphospecific bindingagent is provided, any of a broad range of immunoassays could be readilyadapted to detection of substrate phosphorylated by cMLCK. The abovedescriptions are provided by way of example, and are not intended to belimiting.

EXAMPLE 16 Treatment of Subjects with Compounds That Modulate MyosinPhosporylation

Compounds identified as cMLCK modulators in Example 15 are usefultherapeutically in the treatment of cardiac dysfunction. Such compoundsare, for example, pharmaceuticals, peptides, or antibodies whichactivate or inhibit cMLCK activity.

Compounds may have similarities to compounds that inhibit the smoothmuscle isoform of MLCK. These include, for example, K-252a (J. Biol.Chem. 263: 6215, 1988); the benzothiazolesulfenamide derivativesdescribed in U.S. Pat. No. 5,504,098 and PCT publication WO 9214712;napthalenesulfonamide derivates such as those described by Hidaka etal., Proc. Natl. Acad. Sci. USA, 78: 4354-4357, 1981; amphipathiccalmodulin binding peptides such as those described in U.S. Pat. No.5,840,697; synthetic peptide inhibitors of smooth muscle myosin lightchain kinase, such as those described by Knighton et al., Science 258:130-135, 1992.

Candidate compounds can enhance cMLCK activity. For example, isoforms ofcMLCK with enhanced kinase activity have been identified (see, e.g.,Example 3). Moreover, calmodulin-independent variants of cMLCK(containing the catalytic domain [approximately amino acid residues305-515 in SEQ ID NO: 2], but lacking the calmodulin-binding andautoinhibitory domain [approximately residues 540-590 in SEQ ID NO: 2];see Ikebe et al., Journal of Biological Chemistry. 262: 13828-34, 1987;Pearson et al., Science 241: 970-973, 1988) can readily be constructedand expressed in baculovirus vectors or in eukaryotic expression vectorsor gene therapy vectors. For example, a cDNA encoding amino acidresidues 1-520 of cMLCK expresses a cMLCK variant which isconstitutively active, that is, active in the absence ofcalcium-calmodulin. Expression of such cMLCK variants in the heart of asubject would significantly enhance myosin light chain phosphorylation.

Human subjects harboring cMLCK alleles and mutations described in thisexample, as well as human and non human subjects harboring ELC and RLCmutations, have or are prone to significant cardiac dysfunction. Thedysfunction most commonly observed is that of cardiac hypertrophy anddiastolic dysfunction. The observed associated defects in contractilityare related to stretch activation. The present disclosure reveals thatmyosin phosphorylation decreases the amplitude of stretch activation,thereby diminishing the contribution of stretch activation to overallcardiac contractility. Thus, mutations of cMLCK or RLC that reducemyosin phosphorylation will enhance the contribution of stretchactivation to overall cardiac contractility.

The described mutations that alter myosin phosphorylation point to aintrinsic property which is disclosed herein. The heart tightlycoordinates the set of complex movements it performs in a cardiac cyclethrough myosin phosphorylation. Across the heart, variable levels of RLCphosphorylation diminish or enhance stretch activation in a particularregion of the heart, thereby contributing substantially to the globalfunctioning of the heart. For example, it is revealed here in that theapex of the heart has significantly higher levels of myosinphosphorylation than the papillary muscles and mid ventricular cavity.Such differential phosphorylation enables the apex to contract morerapidly, providing a physiologic explanation for the long-standingsurgeon's observation that the apex moves more rapidly duringcontraction than the mid ventricle or the base.

The described mutations also show that is possible for global orregional alterations in normal myosin phosphorylation to disrupt cardiacfunction, and render the subject susceptible to cardiac hypertrophy,diastolic dysfunction, cardiac failure, and other forms of heartdisease. For example, the cMLCK double mutation ala 87 val, ala 95 glu,results in a cMLCK with increased activity, and is associated with aparticularly virulent form of hereditary cardiac hypertrophy. Thus,inappropriately enhancing myosin phosphorylation leads to (1)inappropriate decreases in the magnitude of stretch activation in one ormore regions of the heart, and (2) inappropriate increases in tension.The result in this particular instance is massive hypertrophy, as theheart seeks to compensate for disrupted stretch activation. Suchpathologic hypertrophy is unfortunately not compensatory, but in factsignificantly worsens the clinical status of the subject.

Even when an obvious phenotype is not associated with a particularmutation, such individuals are believed to have or be prone to cardiacdysfunction. For example, the gly 89 asp cMLCK allele described inExample 3 has an 8% prevalence in African-American populations.African-Americans are known to be disproportionately susceptible tocardiac hypertrophy and diastolic dysfunction, and this predilection canbe explained in part by the high prevalence of the cMLCK gly 89 aspallele (and likely other cMLCK mutations) in this population. Sincehypertrophy and diastolic dysfunction frequently lead to heart failure,modulation of cMLCK activity represents a novel and potential importantnew approach preventing hypertrophy and diastolic dysfunction, therebytreating or preventing heart failure.

Moreover, since heart failure from any cause is characterized bysystolic dysfunction, diastolic dysfunction, or both, regional or globalmodulation of cMLCK activity would be an effective approach to therapyregardless of the cause for cardiac dysfunction. Modulation of stretchactivation is effective even when cardiac dysfunction is notspecifically due to mutations in cMLCK or RLC. For example, by enhancingstretch activation, inhibition of myosin phosphorylation should be anextremely effective approach to cardiac failure. Systolic (contractile)function is significantly enhanced by enhanced stretch activation,whereas diastolic function is also improved, by decreasing thesensitivity of the contractile apparatus to calcium (see Sweeny andStull, American Journal of Physiology 250: C657-660, 1986; Sweeney andStull, PNAS 87: 414-418, 1990).

In one embodiment, an agent identified herein as an effective modulatorof myosin phosphorylation or cMLCK activity (hereinafter “modulator”) isadministered to a subject using techniques well-known in the art. Apharmaceutical composition or cMCLK modulatory peptide or antibody ofthe present invention is combined with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known in the artand include aqueous solutions such as physiologically buffered saline orother buffers or solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize the modulatoror increase the absorption of the modulator. Such physiologicallyacceptable compounds include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients. One skilled in the art would know that thechoice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on the routeof administration and on the particular physio-chemical characteristicsof the specific modulator.

Methods of administering a pharmaceutical are well known in the art. Oneskilled in the art would know that a pharmaceutical compositioncomprising a modulator of the present invention can be administered to asubject by various routes including, for example, orally,intravaginally, rectally, or parenterally, such as intravenously,intramuscularly, subcutaneously, intraorbitally, intracapsularly,intraperitoneally, intracistemally or by passive or facilitatedabsorption through the skin using, for example, a skin patch ortransdermal iontophoresis, respectively. Furthermore, the compositioncan be administered by injection, intubation, orally or topically, thelatter of which can be passive, for example, by direct application of anointment or powder, or active, for example, using a nasal spray orinhalant. A modulator also can be administered as a topical spray, inwhich case one component of the composition is an appropriatepropellant. The pharmaceutical composition also can be incorporated, ifdesired, into liposomes, microspheres or other polymer matrices(Gregoriadis, Liposome Technology, Vol. 1, CRC Press, Boca Raton, Fla.(1984), which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer. Administration can be effectedcontinuously or intermittently and will vary with the subject and isdependent on the type of treatment and potency of the modulator used.

In order to modulate the biological activity of a cMLCK, the modulatormust be administered in an effective dose, which is termed herein as“pharmaceutically effective amount.” The effective dose will, of course,depend on the mode of administration and the relative potency of themodulator. The total effective dose can be administered to a subject asa single dose, either as a bolus or by infusion over a relatively shortperiod of time, or can be administered using a fractionated treatmentprotocol, in which the multiple doses are administered over a moreprolonged period of time. One skilled in the art would know that theconcentration of a cMLCK modulator required to obtain an effective dosein a subject depends on many factors including the age and generalhealth of the subject as well as the route of administration and thenumber of treatments to be administered. In view of these factors, theskilled artisan would adjust the particular dose so as to obtain aneffective dose for modulating cMLCK.

EXAMPLE 17 Genomic Structure of cMLCK

This example presents the twelve exons of cMLCK, together with flankingintronic sequences and primers useful in amplification of segments ofthe cMLCK gene. Exon sequences are presented in ALL CAPS BOLD font.

Examples of sequences which are useful in amplifying a particular exonare underlined. For exons 2 and 3, two primer pairs are provided. Thesense strand is shown in 5′-3′ direction. The primer nearer the 5′ endis synthesized as shown. The primer nearer the 3′ end is synthesized asthe reverse complement of the underlined sequence. For example, toamplify Exon 1 and some flanking intronic sequence, an Exemplary Forwardprimer is: gctagaaagacttgagttagacaa (see SEQ ID NO:3); an ExemplaryReverse primer is: catgcaaacaaggctg (that is, the reverse complement ofcagccttgtttgcatggtgcacg, see SEQ ID NO:3).

For exon 2, the exemplary primers primer pairs are overlapping, with thetwo forward primers being aaaggagggtggatcctgatggtgttctcacctc (see SEQ IDNO:4) and CTGGCCAGGCTAAGATGCAAG (see SEQ ID NO:4). For exon 3, theexemplary primers primer pairs are overlapping, with the two forwardprimers being cctctgtgttctcaccttctag (see SEQ ID NO:5) andCTGGCCAGGCTAAGATGCAAG (see SEQ ID NO:5).

EXON 1 (SEQ ID NO: 3) gactgctcctgagcagccgctggagacagacggcaaccaggttggcccctctttgctccaggtacctctctccccctcagttagcaggctcggcttcctgtctcactgcagccagacgagaggggaaattggacagcctgccacactccactcttgtttctgcagctagaaagacttgagttagacaagcagaagcacacgcctccctacctcATGGCGACAGAAAATGGAGCAGTTGAGCTGGGAATTCAGAACCCATCAACAGgtgccaagctggggcaggagatggagggaggagcttgggaaggggggttttgaatccaggactgggcaaggttccctcagtgggagttctgtgccccagccttgtttgcatggtgcacg EXON 2 (primer pairs overlap, SEQ IDNO: 4) aaaggagggtggatcctgatggtgttctcacctctgcagACAAGGCACCTAAAGGTCCCACAGGTGAAAGACCCCTGGCTGCAGGGAAAGACCCTGGCCCCCCAGACCCAAAGAAAGCTCCGGATCCACCCACCCTGAAGAAAGATGCCAAAGCCCCTGCCTCAGAGAAAGGGGATGGTACCCTGGCCCAACCCTCAACT AGCAGCCAAGGCCCCAAAGGAGAGGGTGACAG GGGCGGGGGGCCCGCGGAGGGCAGTGCTGGGCCCCCGGCAGCCCTGCCCCAGCAGACTGCGACACCTG AGACCAGCGTCAAGAAGCCCAAGGCTGAGCAGGGAGCCTCAG GCAGCCAGGATCCTGGAAAGCCCAGGGTGGGCAAGAAGGCAGCAGAGGGCCAAGCAGCAGCCAGGAGGGGCTCACCTGCCTTTCTGCATAGCCCCAGCTGTCCTGCCATCATCTCCAGgtgaatatcccctcctgggagtggggaggggtcctgtggttctgtccctaggggtcctgcttaattcccttgt EXON 3 (primer pairs overlap, SEQ IDNO: 5) gcgggcttcacctctgtgttctcaccttctag TTCTGAGAAGCTGCTGGCCAAGAAGCCCCCAAGCGAGGCATCAGAGCTCACCTTTGAAGGGGTGCCCATGACCCACAGCCCCACGGATCCCAGGCCAGCCAAGGCAGAAGAAGGAAAG AACATCCTGGCAGAGAGCCAGAAGGAAGTGGGAGAGAAAACCCCAGGCCA GG CTGGCCAGGCTAAGATGCAAGGGGACACCTCGAGGGGGATTGAGTTCCAGGCTGTTCCCTCAGAGAAATCCGAGGTGGGGCAGGCCCTCTGTCTCACAGCCAGGGAGGAGGACTGCTTCCAGATTTTGGgtaggccaggggcaggtgggggctggggctgctctggggccagggggaggaagggggctgtcagtccca agtctacct EXON 4 (SEQID NO: 6) tggtgccaaggggaatcctcagcagcccctggcactgaccatgagggctgtgctctgtcccccagATGATTGCCCGCCACCTCCGGCCCCCTTCCCTCACCGCATGGTGGAGCTGAGGACCGGGAATGTCAGCAGTGAATTCAGTATGAACTCCAAGGAGGCGCTCGGAGGgtgagatctgggaccccagctgggcactc atggacagagagcacaccgEXON 5 (SEQ ID NO: 7) cttggggtcccctaacttacagcctcttctctttccagTGGCAAGTTTGGGGCAGTCTGTACCTGCATGGAGAAAGCCACAGGCCTCAAGCTGGCAGCCAAGGTCATCAAGAAACAGACTCCCAAAGACAAGgtagtgaggttgcgggggtggtggctgcccaggatggggaggggatccttggagtagggcacctctcgcctccctccaccagcagctgctgaacctg EXON 6 (SEQ ID NO: 8)gtaccctttacttccctggtccccagGAAATGGTGTTGCTGGAGATTGAGGTCATGAACCAGCTGAACCACCGCAATCTGATCCAGCTGTATGCAGCCATCGAGACTCCGCATGAGATCGTCCTGTTCATGGAGTAgtgagtggccgaagtagtggtaggggctgggtgggggtaccaccaggcacggagcaagccgtgg a EXON 7 (SEQ ID NO:9) taccaccaggcacggagcaagccgtggaggggtctgtgcacgcaCATCGAGGGCGGAGAGCTCTTCGAGAGGATTGTGGATGAGGACTACCATCTGACCGAGGTGGACACCATGGTGTTTGTCAGGCAGATCTGTGACGGGATCCTCTTCATGCACAAGATGAGGGTTTTGCACCTGGACCTCAAGgtaccagactgggg cctcctgggaag EXON 8(SEQ ID NO: 10) tgcagaggcccacccaggccaccccctttctcctcagCCAGAGAACATCCTGTGTGTCAACACCACCGGGCATTTGGTGAAGATCATTGACTTTGGCCTGGCACGGAGgtaccacctgggtgggtggggagggcaagacaagcctctgag ttggcaggggcaggggtgEXON 9 (SEQ ID NO: 11)ggactgtgctctcagcccttggtctcacccccaggGTATAACCCCAACGAGAAGCTGAAGGTGAACTTTGGGACCCCAGAGTTCCTGTCACCTGAGGTGGTGAATTATGACCAAATCTCCGATAAGACAGACATGTGGAGTATGGGGGTGATCACCTACATGCTgtgagcacccaggagggtcgtgtttatggggttggt EXON 10 (SEQ ID NO:12) cctccaatctcacctccctgccccctgctatcccctccctctagGCTGAGCGGCCTCTCCCCCTTCCTGGGAGATGATGACACAGAGACCCTAAACAACGTTCTATCTGGCAACTGGTACTTTGATGAAGAGACCTTTGAGGCCGTATCAGACGAGGCCAAAGACTTTGTCTCCAACCTCATCGTGAAGGACCAGAGgtgaggctcaccccagaacctgaactgtatgtgtgcaagcttagtgtgtctga gtgctggcagg EXON 11(SEQ ID NO: 13) ccacgtcaccatgctgcctctcccccaGGCCCGGATGAACGCTGCCCAGTGTCTCGCCCATCCCTGGCTCAACAACCTGGCGGAGAAAGCCAAACGCTGTAACCGACGCCTTAAGTCCCAGATCTTGCTTAAGAAATACCTCATGAAGAGGCGCTGGAAGgtaccgctggattcggggtggggagggagggcttgctagtgggaagagctcctggtgccagatcccagc EXON 12 (SEQ ID NO: 14)ccctgccctggtgttgactgggactccctctcttctgccctctagAAAAACTTCATTGCTGTCAGCGCTGCCAACCGCTTCAAGAAGATCAGCAGCTCGGGGGCACTGATGGCTCTGGGGGTCTGAgccctgggcgcagctgaagcctgg acgcagccacacagtgg

EXAMPLE 18 Enzyme Kinetics of the Mutant MLCK

In order to further investigate the effect of the mutant kinase, bothentire wild type and mutant human skeletal/cardiac MLCK proteins (withamino-terminal FLAG® tags) were expressed in baculoviral systems andaffinity purified. Human cardiac RLC, human ventricular ELC and afragment of the β-myosin heavy chain light chain binding region(aa778-840) were co-expressed. Affinity column purification utilizing anexpressed FLAG® tag placed at the carboxy-terminal end of the heavychain fragment yielded a purified complex of the 2 light chains andlight chain binding fragment. This complex was used as substrate in thekinetic studies of the expressed wild type and mutant kinases. A doublereciprocal plot of 1/v vs. 1/[S] comparing the mutant and wild type MLCKwas created. The V_(max) of the mutant is almost double that of thewild-type MLCK (216.±31 vs. 115±15 pmoles min⁻¹ ng⁻¹ respectively),while the K_(m) of the mutant is also significantly greater than thewild type (17.0±4.6 vs. 6.0±1.8 μM respectively). At physiologicconcentrations of light chain (˜300 μM), differences in V_(max) dominatethe rate of RLC phosphorylation.

EXAMPLE 19 Detailed Investigation of Muscle Strain Pattern in LeftVentricle with Non-Invasive Phase-Labeled MRI Techniques (metaDENSE)

A differential pattern of myosin RLC phosphorylation from epicardium toendocardium across the ventricular wall is disclosed herein. This,together with the biophysical changes observed in single fiber studies,predicts an effect on the global pattern of cardiac contraction. Inorder to study the pattern of contraction in the normal human heart,recent developments in phase-labeled MRI motion tracking were utilizedthat allow detailed mapping of muscle strain and torsion distribution inthe left ventricular wall (Aletras et al., 1999; Aletras, 2000;Callaghan, 1991). One such technique, metaDENSE, has been optimized tomap the displacement field of the human heart at 2.8 mm resolutionduring a breath-hold of 14 heartbeats, over the entire systolic ordiastolic period. The spatial resolution of this technique is sufficientto clearly show changes in the contractile strain and torsion of themuscle across the wall. The left ventricle undergoes torsion around itslong axis during systolic contraction, i.e., the apex rotates relativeto the base. Recoil occurs in the diastolic period. The direction oftorsion is consistent with the helical arrangement of the epicardialmuscle fibers but counters the helicity of the endocardial fibers.Therefore, the epicardium is thought to produce the torsion duringsystole.

High resolution torsion measurements with metaDENSE showed 50% to 70%increase in normalized torsion from the epicardial to endocardial border(manuscript in preparation, HW). A color-coded distribution of therotation of the myocardial wall around the LV center in a slice of theleft ventricle perpendicular to its long axis, about one-fourth the LVlength from the apex was generated, derived from a set of metaDENSEimages that encode the wall motion over the entire systolic period. Theendocardium was darker in color than the epicardium, indicating anincrease in the angle of rotation from the epicardial border to theendocardial border. This phenomenon has also been observed with MRtagging studies at lower spatial resolution (Buchalter et al., 1990;Maier et al., 1992; Young et al., 1994).

Having illustrated and described the principles of isolating the humancardiac myosin light chain kinase cDNA and its corresponding genomicgene, the protein and modes of use of these biological molecules, itshould be apparent to one skilled in the art that the invention can bemodified in arrangement and detail without departing from suchprinciples. The scope of the invention is defined by the followingclaims. We therefore claim as our invention all that comes within thescope and spirit of these claims.

1. An isolated antibody or a functional fragment, of the antibody thatspecifically binds a protein consisting of: the amino acid sequence setforth as SEQ ID NO:2.
 2. The isolated antibody or the functionalfragment of the antibody of claim 1, wherein the antibody is amonoclonal antibody.
 3. The isolated antibody or the functional fragmentof the antibody of claim 1, wherein the antibody is a polyclonalantibody.
 4. An isolated antibody or a functional fragment of theantibody that specifically binds a polypeptide consisting of the aminoacid sequence set forth as SEQ ID NO:
 15. 5. The isolated antibody orthe functional fragment of claim 2, wherein the antibody is humanized.6. The isolated antibody or the functional fragment of the isolatedantibody of claim 1, wherein the functional fragment is a Fab fragment,Fab′ fragment, (Fab′)₂ fragment, F(ab′)₂ fragment, Fv or SCA.
 7. Theisolated antibody or the functional fragment of the antibody of claim 1,further comprising a label.
 8. The isolated antibody or the functionalfragment of the antibody of claim 7, wherein the label is a radioactiveisotope, an enzyme substrate, a co-factor, a ligand, a chemiluminescentagent, a fluorescent agent, a hapten or an enzyme.
 9. The isolatedantibody or the functional fragment of the antibody of claim 7, whereinthe label is alkaline phosphatase.
 10. A composition comprising aneffective amount of the antibody or the functional fragment of theantibody of claim 1 and a pharmaceutically acceptable carrier.
 11. Amethod of detecting a cardiac myosin light chain kinase protein in abiological specimen, comprising: (a) contacting a biological specimenwith the antibody or the functional fragment of the antibody of claim 1;and (b) detecting the antibody or the functional fragment of theantibody bound to the biological specimen, thereby detecting the cardiacmyosin light chain kinase protein in the biological specimen.
 12. Themethod of claim 11, wherein the biological specimen comprises cardiacmyocytes.
 13. The method of claim 11, wherein the biological specimencomprises cardiac fibers.
 14. The method of claim 11, further comprisingcomparing cardiac myosin light chain kinase in the biological specimento a control.
 15. The method of claim 11, wherein the antibody or thefunctional fragment of the antibody is labeled.
 16. The method of claim11, wherein detecting antibody bound to the biological specimencomprises: contacting the biological specimen with a second antibodythat specifically binds the antibody or the functional fragment of theantibody bound to the biological specimen, and detecting bound secondantibody, wherein the second antibody comprises a label.
 17. The methodof claim 15, wherein the label is a radioactive isotope, an enzymesubstrate, a co-factor, a ligand, a chemiluminescent agent, afluorescent agent, a hapten or an enzyme.
 18. A method of detecting acardiac myosin light chain kinase protein in a biological specimen,comprising: (a) contacting a biological specimen with the antibody orthe functional fragment of the antibody of claim 4; and (b) detectingthe antibody or the functional fragment of the antibody bound to thebiological specimen, thereby detecting the cardiac myosin light chainkinase protein in the biological specimen.
 19. The method of claim 18,wherein the biological specimen comprises cardiac myocytes.
 20. Themethod of claim 18, wherein the biological specimen comprises cardiacfibers.
 21. The method of claim 18, further comprising comparing cardiacmyosin light chain kinase in the biological specimen to a control. 22.The method of claim 18, wherein the antibody or the functional fragmentof the antibody is labeled.
 23. The method of claim 18, whereindetecting antibody or the functional fragment of the antibody bound tothe biological specimen comprises: contacting the biological specimenwith a second antibody that specifically binds the antibody or thefunctional fragment of the antibody bound to the biological specimen,and detecting bound second antibody, wherein the second antibodycomprises a label.
 24. The method of claim 22, wherein the label is aradioactive isotope, an enzyme substrate, a co-factor, a ligand, achemiluminescent agent, a fluorescent agent, a hapten or an enzyme. 25.The isolated antibody or the functional fragment of the antibody ofclaim 4, wherein the antibody is humanized.
 26. The isolated antibody ofthe functional fragment of the antibody of claim 4, wherein thefunctional fragment is a Fab fragment, Fab′ fragment, (Fab′)₂ fragment,F(ab′)₂ fragment, Fv or SCA.
 27. The isolated antibody or the functionalfragment of the antibody of claim 4, wherein the antibody is amonoclonal antibody.
 28. The isolated antibody or the functionalfragment of the antibody of claim 4, wherein the antibody is apolyclonal antibody.
 29. The isolated antibody or the functionalfragment of the antibody of claim 4, further comprising a label.
 30. Theisolated antibody or the functional fragment of the antibody of claim29, wherein the label is a radioactive isotope, an enzyme substrate, aco-factor, a ligand, a chemiluminescent agent, a fluorescent agent, ahapten or an enzyme.
 31. The isolated antibody or the functionalfragment of the antibody of claim 30, wherein the label is alkalinephosphatase.
 32. A composition comprising an effective amount of theantibody or an effective amount of the functional fragment of theantibody of claim 4 and a pharmaceutically acceptable carrier.